The Duchess of Malfi takes place in Roman Catholic Italy, which English audiences at the time when the play was written would have associated with corruption. It begins in the palace of the Duchess, a young widow and the ruler of the Italian town of Amalfi. Her steward, Antonio, has just returned from a visit to the French court, and Bosola, a murderer and former employee of her brother, the Cardinal, has just returned from his punishment. Soon Duke Ferdinand, the Duchess’s other brother, enters with his whole retinue. In a private conversation with his friend Delio, Antonio reveals that though the Cardinal and Duke appear good, they are in fact jealous, conniving, and despicable. He adds that though her brothers are horrible, the Duchess is noble, temperate, beautiful, and intelligent.
Even though the Duchess is still young and beautiful, her brothers do not want her to remarry. They hope to both preserve their honor by ensuring for ongoing sexual purity, and to eventually inherit her fortune by forcing her to remain a widow. In order to make sure they have their way, Ferdinand gets Bosola a position on the Duchess’s estate and hires him to be his spy. Bosola doesn’t want to be a spy, but he feels that it’s his duty to obey the duke, even if doing so makes him corrupt. Ferdinand and the Cardinal then confront the Duchess with a rehearsed speech instructing her not to remarry. She agrees not to, but as soon as her brothers leave, she tells her maid Cariola that she will marry in secret. The Duchess then woos Antonio, inverting the traditional male and female roles in courtship. The two marry in secret.
Nine months later, the Duchess is pregnant with Antonio’s child. Bosola, still spying for Ferdinand, notes the signs of her pregnancy and plans to give her apricots as a test, because they are known to induce labor. The Duchess eats the apricots and goes into labor, creating chaos in her palace. To try to maintain the secret, Antonio and the Duchess give out a story that the Duchess has fallen ill with some disease. Antonio confronts Bosola to ask if the apricots were poisoned. Bosola denies the accusation, but after the confrontation he notices that Antonio ha accidentally dropped a piece of paper: it is a horoscope for a baby, which provides Bosola with concrete evidence that the Duchess had a child. He decides to send the paper in a letter to the Duchess’s brothers in Rome. When Ferdinand and the Cardinal learn that the Duchess has disobeyed them, they are infuriated, thinking their noble blood has been tainted (and Ferdinand seems also to be overwhelmed with anger by the idea that the Duchess has been having sex at all), but they decide to wait to find out who the father is before taking action.
A few years later, the Duchess has had two more of Antonio’s children. Ferdinand, who learned of the children from Bosola, decides to confront the Duchess in her bedchamber. Ferdinand sneaks in and frightens the Duchess, giving her a knife and suggesting that she kill herself. She admits that she is married, and he becomes enraged. He says that she has lost her reputation, and he swears that he will never see her again in his life. Antonio and the Duchess make a plan to flee: the Duchess announces that Antonio has been using his position to steel from her, and has been fired, as an excuse to get him out of Amalfi. After Bosola privately defends Antonio to the Duchess as being honorable and worthy, the Duchess confides in Bosola that Antonio is innocent and is in fact her husband. She plans to flee to join him.
Back in Rome, the Cardinal and Ferdinand find out from Bosola about the Duchess’s plan. The Cardinal then formally banishes the Duchess, Antonio, and their children. Ferdinand invites Antonio to reconcile, but Antonio believes this is a trap, so instead of accepting the invitation he flees with his eldest son to Milan. After he leaves, Bosola reenters in disguise and takes the Duchess and her other two children captive under orders from the brothers.
The brothers imprison the Duchess in her Amalfi palace. There, because Ferdinand has sworn never to see the Duchess, confronts her in the dark. He gives her his hand to hold, but then reveals that it is the hand of a dead man in order to convince her that Antonio is dead. He then plays a trick with silhouettes to convince the Duchess that her children are also dead, at which point the Duchess wants to die. Ferdinand reveals to Bosola that he plans to torture her by exposing her to madmen from the local insane asylum. While Bosola feels bad for the Duchess and dislikes that he is participating in her torture, he continues to obey the duke.
In her prison, the madmen confront the Duchess and Cariola. Bosola then enters, disguised as an old man, and he tells the Duchess that he’s going to kill her. The Duchess maintains her composure and is unafraid, but executioners enter and strangle her, her two children, and Cariola. Though Ferdinand has no pity for the children, he immediately begins to feel remorse when he sees the Duchess’s dead body. Ferdinand becomes maddened by guilt, and Bosola also acknowledges feeling a guilty conscience. Ferdinand then condemns Bosola for following his orders, and refuses to pay him for his work. After Ferdinand has departed, the Duchess wakes up, but only long enough for Bosola to tell her that her husband is still alive; she dies for real almost immediately after waking up.
Now in Milan, Antonio doesn’t yet know his wife’s fate. He decides to wager everything and confront the Cardinal in person in an attempt to defuse the situation. Ferdinand, meanwhile, has been diagnosed with lycanthropia (werewolf disease), and he begins acting like a madman, even attacking his shadow, clearly plagued by guilt. The Cardinal wants his involvement in the murder to remain secret, and he instructs Bosola to murder Antonio. A woman named Julia, with whom the Cardinal has been having an affair, becomes smitten with Bosola, and he convinces Julia to try and get a confession out of the Cardinal. Julia confronts the Cardinal and finds out his secret, so he forces her to kiss a poisoned book, thereby killing her. Bosola reveals to the Cardinal that he has overseen this murder and the Cardinal’s confession. He agrees to help the Cardinal in return for payment, but in fact this is a trick. He decides that he will do everything in his power to save Antonio and get revenge on the brothers.
In the Cardinal’s palace, the Cardinal tells all of his courtiers to stay away from his room, no matter what they hear, even if he tests them with screams and shouts. Bosola sneaks into the palace, and overhears that the Cardinal plans to kill him after he helps the Cardinal. Soon after, Antonio sneaks into the palace in his effort to find the Cardinal and end their quarrel. However, in the darkness, Bosola accidentally stabs Antonio, mistaking him for one of the brothers. Antonio lives just long enough for Bosola to inform him that the Duchess and two of their children have been murdered, at which point Antonio no longer wants to live. Bosola goes to find and kill the Cardinal, and when the Cardinal starts screaming for help, no one comes because of his instruction that they stay away. In the chaos, Bosola stabs the Cardinal twice. Ferdinand then enters, and, mistaking, his brother for the devil, stabs both the Cardinal and Bosola. Bosola then stabs Ferdinand, who uses his dying words to say that our deaths are caused by our own actions. While Bosola explains what happened, the Cardinal dies, and after Bosola makes a final speech, he dies as well. After all of the deaths, Delio enters with Antonio’s son, announces his intention to help the son to receive his proper inheritance, and ends the play.