In Shakespeare's period, female roles would have been performed by male actors. Some of the comic and ironic effects of his plays thus depend on the audience's awareness of male actors portraying the women in the plays—particularly in the craftsmen's production of Pyramus and Thisbe.

Hermia

Hermia is the daughter of Egeus, an Athenian noble. She is in love with Lysander, but her father wishes her to marry Demetrius instead. When her father tells the duke of her disobedience, Theseus informs her that she must obey Egeus or face the consequences: life in a nunnery or death. Instead, Hermia decides to elope with Lysander and flee Athens. Unfortunately, chaos ensues when Demetrius and Helena follow her and Lysander into the forest. Under the influence of the fairies' magic, Demetrius and Lysander both switch their affections from Hermia to her best friend, Helena, who now becomes her romantic rival. Several of the comic devices during their quarrels reflect their disparity in height, as Hermia is much shorter than Helena. In the end, Hermia is allowed to marry the man of her choosing: Lysander.

Helena

Also a young woman of a noble Athenian family, Helena is in love with Demetrius. Though they were betrothed, the fickle Demetrius deserted Helena to court her friend Hermia—a match Hermia's father approves of. When Hermia and Lysander run away together, Helena alerts Demetrius—thinking that this may help her win back his love—and they follow the eloping couple into the forest. Oberon witnesses Demetrius's rejection of Helena, and pitying her, he orders Puck to give Demetrius a love potion that will make him fall for Helena. Puck accidentally gives the potion to Lysander, and then Demetrius, leading both men to declare their love for Helena. Taken aback by this turn of events, and perhaps lacking in self-confidence, Helena is convinced the two men are playing a cruel trick on her. Puck eventually fixes his mistake by restoring the bond between Hermia and Lysander, leaving Demetrius in love with Helena.

Lysander

Lysander is a young man in love with Hermia. Although Hermia's father, Egeus, would prefer her to marry Demetrius, Lysander does not relent. When pleading their case to Thesus fails to work, Lysander suggests to Hermia that they run away to his aunt's house. Since she dwells outside of Athens, they will be free to from the duke's rule and may marry. Their plan goes awry, however, when Puck accidentally gives a magic potion to Lysander (believing him to be Demetrius). The magic causes Lysander to temporarily fall in love with Hermia's friend Helena—much to Hermia's confusion and dismay. Eventually, the effects of the potion are undone, and Lysander goes back to loving Hermia, thinking his strange adventure in the woods a dream. At the end of the play, the duke changes his mind, and allows Lysander and Hermia to marry.

Demetrius

A somewhat fickle young man, Demetrius was initially in love with—and betrothed to—Helena but switched his affections to Hermia upon seeing her for the first time. With the support of Egeus, Demetrius pursues Hermia relentlessly at the beginning of the play, even though she has openly declared her love for Lysander. Demetrius even follows them into the forest when they attempt to elope. In the chaos of the woods, Demetrius is given a potion that leads him to fall in love with Helena. Unlike Lysander, who has the effects of the love potion reversed, Demetrius remains in love with Helena, which allows both couples to find happiness.

Theseus

Theseus is a legendary duke of Athens. When the play opens, he is preparing to marry Hippolyta, the former queen of the Amazons. He is known in legend for his wisdom and sense of justice. Initially, he sides with Egeus in the dispute over whom Hermia should marry, but by the end of the play, he has changed his mind and allows her to wed Lysander. It is for his spectacular wedding feast that the craftsmen are preparing their play.

Hippolyta

In Greek mythology (and this play), Hippolyta was the queen of the Amazons. She is captured by Theseus and forced to become his wife. Shakespeare portrays her as eventually willing to marry Theseus rather than as a victim of rape, which was the case in earlier portrayals of her.

Egeus

Egeus is an Athenian noble and Hermia's father. He wishes his daughter to make an advantageous marriage to Demetrius. When Hermia refuses, on account of her love for Lysander, Egeus involves the duke and implores him to enforce the law, which states that Hermia must obey him or suffer dire consequences.

Oberon

Oberon is the king of the fairies and a being with magical powers. He is feuding with his wife, Titania, over a changeling boy, whom he wants for a knight. During their feud, he uses magic to make Titania fall in love with Bottom, but he later restores her to her senses. It is Oberon who orders Puck to enchant the feuding lovers, and chaos ensues when Puck gives a love potion to the wrong Athenian man.

Titania

The queen of the fairies, Titania is beautiful and regal. She refuses to relinquish a changeling boy to her husband, and their feud over the boy leads her husband to give her a love potion. Under the influence of the potion, she becomes infatuated with Bottom, whom Puck has enchanted to have the head of an ass. After Oberon claims the changeling boy, he restores her to her senses.

Puck

Puck, or Robin Goodfellow, is a fairy with magical powers who serves Oberon. He is a figure from English folk legend, mischievous but not malevolent. He is a loyal servant but is liable to misinterpreting instructions, and he likes to have a bit of fun of his own while he's carrying out Oberon's missions (for example, turning Bottom's head into that of an ass was Puck's idea).

Nick Bottom

Bottom is a weaver who will be performing in Quince's play at Theseus's wedding. He is a "mechanical," a lower-class figure introduced for comic effect. Puck gives him the head of a donkey and makes Titania fall in love with him. Bottom is somewhat confused by this but retains his cheerful nature. Bottom's extreme self-confidence, despite his foolish ideas and manner, serves as a point of humor throughout the play—particularly when he is paired with the elegant Titania, who has been enchanted into thinking she loves him.

Peter Quince

Peter Quince, a carpenter, is the director of the bumbling troop of craftsmen ("mechanicals") rehearsing the play to present at the wedding of Theseus. Though he is the director, he is often overshadowed by the Bottom, who confidently offers unsolicited and silly advice on how the play should be performed.

Francis Flute

A bellows-mender by occupation, Flute is cast as Thisbe in the craftsmen's production of Pyramus and Thisbe. Flute isn't pleased to have been cast in the role of woman, and he decides that he should adopt a falsetto voice for his speaking parts.

Snug

Snug is a joiner who is cast as the lion in the craftsmen's play. He worries about remembering his lines, until he's informed that his only job is to roar. The role of the lion is later revised to include a disclaimer that Snug is not, in fact, a lion—added due to the craftsmen's fear that his portrayal of a lion might frighten any ladies in the audience.

Robin Starveling

Starveling is a tailor. He starts out in the role of Thisbe's mother but ends up with the role of Moonshine.

Tom Snout

A tinker, Snout is originally cast as Pyramus's father but is later given the part of Wall, where he pretends to be the literal wall that separates the two lovers in the play.

Philostrate

Philostrate is Thesus's master of revels and, in some versions of the play, is the one who offers Theseus the list of performances to choose from (one of which is the craftsmen's play).

Titania's Fairies

Cobweb, Mote, Mustardseed, and Peaseblossom are the fairies who serve Titania. She orders them to wait on Bottom when she is under the love spell.