Mr. Allwit
Mr. Allwit knowingly lets his wife have an affair with Sir Walter Whorehound and in return, Sir Walter covers all of the Allwits' living expenses. Allwit is an example of a willing cuckold known as a wittol. For Allwit and for his wife, their marriage is more like a business arrangement than a traditional, romantic marriage. Allwit allows Sir Walter to be his wife's lover to the point where Allwit has lost the privilege of sleeping with his wife at all, as a scene with the jealous Sir Walter indicates. Allwit is also suspicious of Sir Walter, cautious that his benefactor may someday try to marry and no longer need Allwit's wife. When Allwit realizes that Sir Walter has come to town to marry Moll Yellow-hammer, he tries to stop it by telling Mr. Yellow-hammer that Sir Walter has had mistresses. Despite their strange arrangement, Allwit does genuinely enjoy his children—all of whom are bastards fathered by Sir Walter. When Sir Walter seeks redemption at the end of the play, thinking he is mortally wounded, Allwit tries to comfort him by bringing in two of these bastard children—Wat and Nick. However, when Sir Walter says that he will leave Allwit and his wife only curses in his will and when Allwit hears Sir Walter has killed a man and is a wanted fugitive, Allwit suddenly changes his tune and no longer wants anything to do with Sir Walter. He refuses Sir Walter sanctuary, and Allwit and his wife decide to use the possessions bought for them by Sir Walter to outfit a house in the Strand—the fashionable part of London.

Mrs. Allwit
With her husband's knowledge, Mrs. Allwit has an affair with Sir Walter Whorehound. In return, Sir Walter covers all of the Allwits' living expenses. Mrs. Allwit's marriage to her husband is more like a business arrangement than a romantic marriage. When the play begins, Mrs. Allwit is about to give birth to her latest child by Sir Walter. This fact and the event of the new baby's christening, give the play some of the most humorously ironic scenes—a fact noted by many critics. When Sir Walter seeks redemption at the end of the play, thinking he is mortally wounded, Allwit tries to comfort him by bringing in some of the bastard children that Mrs. Allwit has had by Sir Walter. This only makes Sir Walter more distressed, and he accuses Mrs. Allwit of helping to damn his soul by being his mistress. When Allwit tries to throw Sir Walter out after it is revealed that Sir Walter is a fugitive, Mrs. Allwit tries to intervene on Sir Walter's behalf at first. Ultimately, Mrs. Allwit sides with her husband. After they kick out Sir Walter, it is Mrs. Allwit who suggests they use their extra possessions to secure a house in the Strand.

Davy Dahanna
Davy Dahanna is Sir Walter Whorehound's poor relative and personal servant. Throughout the play, Dahanna makes many humorous asides to the audience at the expense of Sir Walter and others. Dahanna is the one who notifies Mr. Allwit of Sir Walter's impending marriage. Dahanna is hoping that if Allwit can stop the marriage and Sir Walter dies childless, Dahanna may gain the inheritance from his distant relation, Sir Kix.

Mrs. Kix
Mrs. Kix, wife of Sir Kix, is distraught that they cannot conceive a child so she gets pregnant by Touchwood Senior. The Kixes are related to Sir Walter in an unspecified way, but the play does indicate that if the Kixes do not bear an heir, they will lose their fortune to Sir Walter. For this reason, the Kixes' childless state becomes a source of strife between them, and Mrs. Kix blames her husband, saying that she never had fertility problems before. After Mrs. Kix and her husband learn of the special fertility drink that Touchwood Senior can sell them, Mrs. Kix encourages her husband to buy it. While her husband drinks the elixir and is sent off on a long horseback ride—which Touchwood Senior says is the only way to make the drink work—the extremely fertile Touchwood Senior impregnates Mrs. Kix in her coach.

Sir Oliver Kix
Sir Oliver Kix is related to Sir Walter and the two are in competition to see who can produce the first legitimate heir and thus secure the Kixes' fortune; Kix unwittingly allows himself to be cuckolded. The Kixes' childless state becomes a source of strife between them, and Sir Kix blames his wife, saying she is barren—even though Sir Kix is an old man and is more likely culpable for their sterile condition. When Sir Kix hears about Touchwood Senior's fertility drink, he buys a vial of the drink from the latter. However, Touchwood Senior tells Sir Kix that in order for the drink to work, Sir Kix must take a long horseback ride to properly mix up the elixir. Sir Kix falls for this deception and, while he is gone on his trip, the extremely fertile Touchwood Senior impregnates Mrs. Kix. Sir Kix is so happy when his wife conceives that he offers to feed and house Touchwood Senior and all of his children including any other children that Touchwood Senior might have in the future.

Touchwood, Junior
Touchwood Junior is in love with Moll Yellowhammer, but her parents forbid her to marry him. The lovers try to marry in secret, but they are caught before they can be wed. Next, they try to run away together across the river, but Maudlin Yellow-hammer jumps in the river, catches Moll, and drags her out. Finally, the two lovers fake their deaths. For Touchwood Junior, he fakes his by acting like he is mortally wounded in a duel with Sir Walter Whorehound. However, at the funeral for the lovers, Touchwood Junior and Moll rise up out of their coffins and get married before the Yellowhammers can stop them.

Touchwood, Senior
Touchwood Senior is an extremely fertile man who has more children than he can support and as a result, he and his wife plan to live apart before they have any more children. Touchwood Senior finds a benefactor for his children when he sells a fake elixir to Sir Oliver Kix, saying that it will make him fertile enough to impregnate his wife. However, while Kix is off taking the elixir, Touchwood Senior impregnates Mrs. Kix himself. Sir Kix is so happy that his wife is pregnant that he agrees to support Touchwood Senior and his family.

The Welsh Gentlewoman
The Welsh Gentlewoman is a prostitute whom Sir Walter Whorehound poses as his niece, who is to be married to Tim Yellowhammer in return for Sir Walter getting to marry Moll Yellowhammer. The Welsh Gentlewoman is married to Tim at the same time as the supposed funeral of Moll Yellowhammer. Although Tim is distraught when he finds out he has married a prostitute, she tells him that marriage makes her honest.

Maudlin Yellowhammer
Maudlin Yellowhammer tries to help her husband force their daughter, Moll, to marry Sir Walter Whorehound as an exchange for having their son marry the Welsh Gentlewoman, whom they believe is rich. Although Moll is one of the few chaste characters in the play, Maudlin treats Moll like she is worthless especially when Moll tries on two separate occasions to escape her marriage to Sir Walter. On the second occasion, Maudlin literally drags Moll out of the river by her hair. When Moll uses this incident to fake her own death, Moll's parents mourn her loss but do not attend the funeral because they are too busy trying to marry their son Tim to the Welsh Gentlewoman before Sir Walter Whorehound finds out that his intended bride is dead. As a result, the Yellowhammers arrive too late to Moll's funeral—where Moll and Touchwood Junior reveal that they are alive—and are unable to stop Moll and Touchwood Junior from marrying. Moll Yellowhammer
Moll is the chaste maid of the play's title who wishes to marry Touchwood Junior, but her parents try to force her to marry Sir Walter Whorehound instead. Moll and Touchwood Junior try to marry in secret, but they are caught before they can be wed. Next, they try to run away together across the river, but Maudlin Yellowhammer jumps in the river, catches Moll and drags her out. Moll uses the incident to fake her death by acting as if she caught ill when her mother pulled her out of the river. At the same time, Touchwood Junior fakes his death by acting like he was mortally wounded in a duel with Sir Walter Whorehound. At the funeral for the lovers, Moll and Touchwood Junior rise up out of their coffins and get married before the Yellowham-mers can stop them.

Mr. Yellowhammer
Yellowhammer is a goldsmith who along with his wife, Maudlin, tries to force his daughter, Moll, to marry Sir Walter Whorehound. Yellowhammer is blind to the fact that Touchwood Junior is having him make a wedding ring with which to steal Moll. However, Yellowhammer arrives in time to stop the two lovers' first attempt at marriage. When Allwit comes to see Yellowhammer, he poses as a relative who is trying to give Yellowhammer some advice. He tells Yellowhammer that Sir Walter is an adulterer and Yellowhammer tells Allwit that he will not have Sir Walter marry his daughter. However, when Allwit is gone, Yellowhammer notes that he himself has kept mistresses before, and that he still plans on going through with the marriage of Moll and Sir Walter. When Moll fakes her death, her parents mourn her loss but do not attend the funeral because they are too busy trying to marry their son Tim to the Welsh Gentlewoman before Sir Walter Whorehound finds out that his intended bride is dead. As a result, the Yellow hammers arrive too late to Moll's funeral—where Moll and Touchwood Junior reveal that they are alive—and are unable to stop Moll and Touchwood Junior from marrying. However, Yellowhammer realizes that it could be worse, as he has just married his son to a prostitute and finds comfort in the fact that he only has to pay for one dinner to serve both of his children's weddings.

Tim Yellowhammer
Tim is a university student who is unwittingly led to marry a prostitute posing as the Welsh Gentlewoman. Tim is very dim-witted, but he thinks that his university education makes him smart. As a result, he tries to use Latin whenever he can, much to the dismay of his mother and the Welsh Gentlewoman who thinks he is insulting her. Tim thinks that logic can solve anything and spends much of his time locked in logical debates with his tutor. Although at first he is apprehensive about his intended union with the Welsh Gentlewoman, he grows to be very fond of her. When he finds out after they are married that she is a prostitute, he is distraught until his mother reminds him that he once said he could use logic to prove a prostitute to be an honest woman— and now he has his chance. Ultimately, Tim accepts his wife when she says that, despite her past, marriage makes her an honest woman.