The play is set in the court of Malfi (Amalfi), Italy 1504 to 1510. The recently widowed Duchess falls in love with Antonio, a lowly steward, but her brothers, not wishing her to share their inheritance and desperate to evade a degrading association with their social inferiors, forbid her from remarrying. She marries Antonio in secret and bears him three children before they are found out.
The Duchess's lunatic and incestuously obsessed brother, Ferdinand threatens and disowns her. In an attempt to escape, she and Antonio concoct a story that he has swindled her out of her fortune and has to flee into exile. She takes Bosola into her confidence, not knowing that he is Ferdinand's spy, and arranges that he will deliver her jewellery to Antonio at his hiding-place in Ancona. She will join them later, whilst pretending to make a pilgrimage to a town nearby. The Cardinal hears of the plan, instructs Bosola to banish the two lovers, and sends soldiers to capture them. Antonio escapes with their eldest son, but the Duchess, her maid, and her two younger children are returned to Malfi and, under instructions from Ferdinand, die at the hands of Bosola's executioners. This experience, combined with a long-standing sense of injustice and his own feeling of a lack of identity, turns Bosola against the Cardinal and his brother, deciding to take up the cause of "Revenge for the Duchess of Malfi" (V.2).
The Cardinal confesses to his mistress, Julia, his part in the killing of the Duchess and then murders her to silence her, using a poisoned Bible. Next, Bosola overhears the Cardinal plotting to kill him (though he accepts what he sees as punishment for his actions) and so visits the darkened chapel to kill the Cardinal at his prayers. Instead, he mistakenly kills Antonio, who has just returned to Malfi to attempt a reconciliation with the Cardinal. Bosola then stabs the Cardinal, who dies. In the brawl that follows, Ferdinand and Bosola stab each other to death.
Antonio's elder son by the Duchess appears in the final scene and takes his place as the heir to the Malfi fortune, despite his father's explicit wish that he "fly the court of princes", a corrupt and increasingly deadly environment.