The play opens with a Prologue narrated by Machevill, a caricature of the author Machiavelli. This character explains that he is presenting the "tragedy of a Jew" who has become rich by following Machiavelli's teachings.
Act I opens with a Jewish merchant, called Barabas, waiting for news about the return of his ships from the east. He discovers that they have safely docked in Malta, before three Jews arrive to inform him that they must go to the senate-house to meet the governor. Once there, Barabas discovers that along with every other Jew on the island he must forfeit half of his estate to help the government pay tribute to the Turks. When the Barabas protests at this unfair treatment, the governor Ferneze confiscates all of Barabas's wealth and decides to turn Barabas's house into a convent. Barabas vows revenge but first attempts to recover some of the treasures he has hidden in his mansion. His daughter, Abigail, pretends to convert to Christianity in order to enter the convent. She smuggles out her father's gold at night.
Ferneze meets with Del Bosco, the Spanish Vice-Admiral, who wishes to sell Turkish slaves in the market place. Del Bosco convinces Ferneze to break his alliance with the Turks in return for Spanish protection. While viewing the slaves, Barabas meets up with Ferneze's, Lodowick. This man has heard of Abigail's great beauty from his friend (and Abigail's lover) Mathias. Barabas realizes that he can use Lodowick to exact revenge on Ferneze, and so he dupes the young man into thinking Abigail will marry him. While doing this, the merchant buys a slave called Ithamore who hates Christians as much as his new master does. Mathias sees Barabas talking to Lodowick and demands to know whether they are discussing Abigail. Barabas lies to Mathias, and so Barabas deludes both young men into thinking that Abigail has been promised to them. At home, Barabas orders his reluctant daughter to get betrothed to Lodowick. At the end of the second Act, the two young men vow revenge on each other for attempting to woo Abigail behind one another's backs. Barabas seizes on this opportunity and gets Ithamore to deliver a forged letter to Mathias, supposedly from Lodowick, challenging him to a duel.
Act III introduces the prostitute Bellamira and her pimp Pilia-Borza, who decide that they will steal some of Barabas's gold since business has been slack. Ithamore enters and instantly falls in love with Bellamira. Mathias and Lodowick kill each other in the duel orchestrated by Barabas and are found by Ferneze and Katherine, Mathias's mother. The bereaved parents vow revenge on the perpetrator of their sons' murders. Abigail finds Ithamore laughing, and Ithamore tells her of Barabas's role in the young men's deaths. Grief-stricken, Abigail persuades a Dominican friar Jacomo to let her enter the convent, even though she lied once before about converting. When Barabas finds out what Abigail has done, he is enraged, and he decides to poison some rice and send it to the nuns. He instructs Ithamore to deliver the food. In the next scene, Ferneze meets a Turkish emissary, and Ferneze explains that he will not pay the required tribute. The Turk leaves, stating that his leader Calymath will attack the island.
Jacomo and another friar Bernardine despair at the deaths of all the nuns, who have been poisoned by Barabas. Abigail enters, close to death, and confesses her father's role in Mathias's and Lodowick's deaths to Jacomo. She knows that the priest cannot make this knowledge public because it was revealed to him in confession.
Act IV shows Barabas and Ithamore delighting in the nuns' deaths. Bernardine and Jacomo enter with the intention of confronting Barabas. Barabas realizes that Abigail has confessed his crimes to Jacomo. In order to distract the two priests from their task, Barabas pretends that he wants to convert to Christianity and give all his money to whichever monastery he joins. Jacomo and Bernardine start fighting in order to get the Jew to join their own religious houses. Barabas hatches a plan and tricks Bernardine into coming home with him. Ithamore then strangles Bernardine, and Barabas frames Jacomo for the crime. The action switches to Bellamira and her pimp, who find Ithamore and persuade him to bribe Barabas. The slave confesses his master's crimes to Bellamira, who decides to report them to the governor after Barabas has given her his money. Barabas is maddened by the slave's treachery and turns up at Bellamira's home disguised as a French lute player. Barabas then poisons all three conspirators with the use of a poisoned flower.
The action moves quickly in the final act. Bellamira and Pilia-Borza confess Barabas's crimes to Ferneze, and the murderer is sent for along with Ithamore. Shortly after, Bellamira, Pilia-Borza and Ithamore die. Barabas fakes his own death and escapes to find Calymath. Barabas tells the Turkish leader how best to storm the town. Following this event and the capture of Malta by the Turkish forces, Barabas is made governor, and Calymath prepares to leave. However, fearing for his own life and the security of his office, Barabas sends for Ferneze. Barabas tells him that he will free Malta from Turkish rule and kill Calymath in exchange for a large amount of money. Ferneze agrees and Barabas invites Calymath to a feast at his home. However, when Calymath arrives, Ferneze prevents Barabas from killing him. Ferneze and Calymath watch as Barabas dies in a cauldron that Barabas had prepared for Calymath. Ferneze tells the Turkish leader that he will be a prisoner in Malta until the Ottoman Emperor agrees to free the island.
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