The play is set in an unnamed Italian court.
Vindice broods over his father's recent death and his desire for revenge on the lustful Duke for poisoning his beloved nine years before. Vindice's brother Hippolito brings news: Lussurioso, the Duke's heir by his first marriage, has asked him if he can find a procurer to obtain a young virgin he lusts after; the brothers decide that Vindice will undertake this role in disguise, to give an opportunity for their revenge. Meanwhile Lord Antonio's wife has been raped by the new Duchess's youngest son (Junior Brother). He brazenly admits his guilt, even joking about it, but to widespread surprise the Duke suspends the proceedings and defers the court's judgement. The duchess's other sons, (Ambitioso and Supervacuo) whisper a promise to have him freed; the Duchess vows to be unfaithful to the Duke. Spurio (the Duke's bastard son) agrees to be her lover but, when alone, declares he hates her and her sons as intensely as he hates the Duke and Lussurioso. Vindice, disguised as 'Piato', is accepted by Lussurioso, who tells him that the virgin he desires is Vindice's sister, Castiza; and he predicts her mother will accept a bribe and be a 'bawd to her own daughter'. Vindice, alone, vows to kill Lussurioso, but decides meanwhile to stay in disguise and put his mother and sister to the test by tempting them. Antonio's wife commits suicide; Antonio displays her dead body to fellow mourners and Hippolito swears all those present to revenge her death.
Vindice, disguised as 'Piato', tests the virtue of his sister and mother. Castiza proves resolute but his mother yields to an offer of gold. Vindice gives Lussurioso the false news that Castiza's resistance to his advance is crumbling. Lussurioso resolves he must sleep with her that same night. Hippolito and Vindice by chance overhear a servant tell Spurio that Lussurioso intends to sleep with Castiza 'within this hour'. Spurio rushes away to kill Lussurioso in flagrante delicto. A moment later Lussurioso himself enters, on his way to Castiza, but Vindice deceptively warns him that Spurio is bedding the Duchess. Angered, Lussurioso rushes off to find Spurio, and bursts into the ducal bedchamber, only to find his father lawfully in bed with the Duchess. Lussurioso is arrested for attempting treason; in the excitement Hippolito and Vindice discreetly withdraw. The Duke, seeing through Ambitioso and Supervacuo's pretended reluctance to see Lussurioso executed, dispatches them with a warrant for the execution of his son 'ere many days' – but once they have gone he gives a countermanding order for his son's release.
Ambitioso and Supervacuo set off directly to the prison to order the instant execution of Lussurioso. Before they arrive however (and unknown to them) the Duke's countermanding order is obeyed – Lussurioso is freed. Ambitioso and Supervacuo arrive at the prison and present the Duke's first warrant to execute (in their words) 'our brother the duke's son'. The guards misinterpret these words, taking instead the youngest son out to instant execution. Meanwhile Vindice is hired again as a pandar – this time by the Duke himself. His plan is to procure the Duke an unusual lady – a richly clothed effigy, her head the skull of Vindice's beloved and covered with poison. The meeting is in a dark and secret place near where the Duchess has arranged a meeting with Spurio. The Duke is poisoned in kissing the supposed lady and is subsequently stabbed by Vindice after being forced to watch the Duchess betray him with Spurio. Ambitioso and Supervacuo, still confident that Lussurioso has been executed, both look forward to succeeding the throne in his place. A freshly severed head is brought in from prison. Assuming it is Lussurioso's, they are gloating over it when Lussurioso himself arrives, alive. They realise to their dismay the head is the youngest son's.
Lussurioso tells Hippolito he wants to get rid of 'Piato', and asks if Vindice (of whom he knows only by report) would replace him. Hippolito assents, realising that Lussurioso would not recognise Vindice without a disguise. Vindice gets his new mission – to kill 'Piato'. Hippolito and Vindice take the corpse of the Duke and dress it in 'Piato's' clothes, so that when the corpse is found it will be assumed that 'Piato' murdered the Duke then switched clothes with him to escape. Vindice and Hippolito confront Gratiana for her earlier willingness to prostitute Castiza, and bring her to repentance.
The scheme with the Duke's corpse is successful, and the Duke's death becomes public knowledge. Vindice and Hippolito lead a group of conspirators which, shortly after the installation of Lussurioso as Duke, kills the new Duke and his supporters. A second group of murderers, including Supervacuo, Ambitioso and Spurio, then arrives; they discover their intended victims already dead, and then turn on and kill each other. The dying Lussurioso is unable to expose Vindice's treacheries to Lord Antonio. Exhilarated by his success and revenge, Vindice confides in Antonio that he and his brother murdered the old Duke. Antonio, appalled, condemns them to execution. Vindice, in a final speech, accepts his death.