The Duchess of Malfi is a young widow whose two brothers, a cardinal and Ferdinand, the duke of Calabria, are desperately anxious lest she marry again, for they want to inherit her title and her estates. Their spy in her household is Bosola, her master of horse.

The duchess falls in love with her steward, Antonio, and marries him secretly. Later, she secretly bears a son. When the happy father writes out the child’s horoscope according to the rules of astrology and then loses the paper, Bosola finds the document and learns about the child. He dispatches a letter immediately to Rome to inform the brothers. The duke swears that only her blood can quench his anger, and he threatens that once he knows the identity of the duchess’s lover, he will ruin her completely.

The years pass and the duchess bears Antonio two more children, a second son and a daughter. Antonio tells his friend Delio that he is worried because Duke Ferdinand is too quiet about the matter and because the people of Malfi, not aware of their duchess’s marriage, are calling her a common strumpet.

Duke Ferdinand comes to the court to propose Count Malateste as a second husband for the duchess. She refuses. Bosola is not able to discover the father of the duchess’s children. Impatient with his informer, the duke decides on a bolder course of action. He determines to gain entrance to the duchess’s private chamber and there to wring a confession from her. That night, using a key Bosola gives him, the duke goes to her bedroom. Under his threats, she confesses to her second marriage, but she refuses to reveal Antonio’s name. After the duke leaves, she calls Antonio and her loyal servant Cariola to her chamber. They plan Antonio’s escape from Malfi before his identity can become known to the duchess’s brothers.

The duchess calls Bosola and tells him that Antonio falsified some accounts. As soon as Bosola leaves, she recalls Antonio and tells him of the feigned crime of which she accused him to shield both their honors, and then bids him flee to the town of Ancona, where they will meet later. In the presence of Bosola and the officers of her guard she accuses Antonio of stealing money and banishes him from Malfi. With feigned indignation, Antonio replies that such is the treatment of thankless masters, and he leaves for Ancona. When the duped Bosola upholds Antonio in an argument with the duchess, she feels she can trust him with the secret of her marriage and asks him to take jewels and money to her husband at Ancona. Bosola, in return, advises her to make her own departure from the court more seemly by going to Ancona by way of the shrine of Loretto, so that the flight might look like a religious pilgrimage.

Bosola immediately travels to Rome, where he betrays the plans of Antonio and the duchess to Duke Ferdinand and the cardinal. They thereupon promptly have the lovers banished from Ancona. Bosola meets the duchess and Antonio near Loretto with a letter from Duke Ferdinand that orders Antonio to report to him, since he now knows Antonio to be his sister’s husband. Antonio refuses and flees with his older son toward Milan. Bosola takes the duchess back to her palace at Malfi as Duke Ferdinand’s prisoner. At Malfi, the duke again visits her in her chamber. He presents her with a dead man’s hand, implying that it is from Antonio’s corpse. Finally Bosola comes and strangles the duchess. Cariola and the children are also strangled, though not with the quiet dignity with which the duchess accepted her fate. When Bosola asks Duke Ferdinand for his reward, the hypocritical duke laughs and replies that the only reward for such a crime is its pardon.

In Milan, meanwhile, Antonio plans to visit the cardinal’s chamber during the night to seek a reconciliation with the duchess’s brothers. He intends to approach the cardinal because Duke Ferdinand loses his mind after causing his sister’s murder. The cardinal orders Bosola that same evening to seek out Antonio, who is known to be in Milan, and murder him, but Bosola turns on him and accuses him of having plotted the duchess’s murder. He demands his reward. When a reward is again refused, Bosola decides to join forces with Antonio to avenge the duchess’s death.

That night, all plans miscarry. In the dark, Bosola accidentally murders Antonio, the man he hoped to make an ally in his revenge on Duke Ferdinand and the cardinal. A few minutes later, Bosola stabs the cardinal and is in turn stabbed by the mad Duke Ferdinand, who rushes into the room. Bosola, with his last strength, stabs the duke and they both die. Alarmed, the guards break into the apartments to discover the bodies. Into the welter of blood, a courtier leads the younger son of the Duchess of Malfi and Antonio, whom Antonio took to Milan. He is proclaimed ruler of the lands held by his mother and his uncles.