Merchants and Jews visit Barabas in his counting house. Through their dialogue they reveal the play's background: The Turks have allowed the Maltese tribute to accumulate for years. Suddenly they want the entire tribute paid at once. They send Selim-Calymath to collect it from the Maltese Governor, Ferneze. The Governor asks for one month to raise the tribute. Selim-Calymath agrees to the delay.

The Governor commands the Jews to yield up half of their wealth to the city. Failing that, they are to renounce their faith and become Christians or, in the alternative, suffer the loss of all of their wealth to the state. Barabas refuses the order and loses his great wealth; his house is confiscated and turned into a convent.

Barabas has hidden a large sum of money in gold under the floor of his house, and he convinces his daughter, Abigail, to pretend to convert to Christianity so that she may, under the guise of taking orders, enter the convent as a novice and remove the money. This she does, drops the money from the balcony at night to the awaiting Barabas, renounces her desire to enter the convent, and returns to the bosom of her father.

Newly enriched, Barabas buys a Turkish slave, Ithamore, who, like Barabas, is a Machiavel and lover of evil doing. The master and slave conspire to avenge Barabas's wrongs on the Governor's son, Lodowick.

Lodowick, who is enamored of Abigail, is invited to the Jew's house and told he is Barabas's favorite for the hand of his daughter. Abigail meanwhile is in love with Mathias, an upright and honorable young man and friend to Lodowick. Barabas convinces Abigail to act as if she loves Lodowick (but only for Lodowick's sake, because she may still in fact love Mathias—and Abigail agrees to the wishes of her father, unaware of his darker intentions). Barabas secretly tells Mathias that Mathias is Barabas's favorite for the hand of Abigail. Barabas arranges everything to look as though the two men are rivals and convinces each that the other has intentions of taking Abigail against Barabas's will. Mathias and Lodowick fall into a duel in which they kill one another.

Abigail, horrified by Barabas's act of killing her beloved, goes to a friar and seeks admission into the convent again, this time in earnest. The friar (#1) allows her again to prepare to take her orders and become a novice in the nunnery. Barabas, angered by his daughter's desertion and her renunciation of Judaism, and fearful that she might betray his evil deed, sends a pot of poisoned rice porridge to the convent. All the nuns eat the porridge, including Abigail, and die.

Before her death, however, Abigail confesses to friar (#2) her complicity in the deaths of Lodowick and Mathias, and of her suspicions of Barabas's hand in poisoning the convent. She does this trusting in the confidentiality of confessions, believing she has not betrayed or endangered her father. She dies.

Friars #1 and #2, each from different orders, rush to Barabas and threaten to betray him to the law. Barabas confesses both his actions and his willingness to enter the priesthood, giving his vast wealth to whichever order he enters. The friars each try to convince him to enter his own order, and Barabas—as he did with Lodowick and Mathias—manages to convince each that he favors his order and rejects the other. He asks one to come at suppertime and the other to come some little time later. He strangles the first friar at supper, props him up on his staff at the entrance to his home. The second friar accosts the corpse, believing it to be the first friar barring him entrance to Barabas. He strikes the corpse to the ground. Barabas emerges and accuses the second friar of killing the first.

The second friar is hanged for the crime. Ithamore, the Turkish slave, becomes enamored of a Courtesan, who, along with her procurer (Pilia-Borza) convinces Ithamore to blackmail Barabas for money. Barabas disguises himself as a minstrel and comes to Ithamore, the Courtesan, and Pilia-Borza with a poisoned bouquet of flowers.

In the meantime the Governor has secured the promise of military aid from Spain and decides not to turn over the tribute to the Turks. He tells Selim-Calymath as much and earns the promise of Turkish reprisals.

The trio of blackmailers (Ithamore, Courtesan, and Pilia-Borza) turns states evidence on Barabas and all four are led away by the Governor's men. The trio succumbs to the poisoned bouquet and die.

Barabas, having drunk a mixture of poppy and cold mandrake juice, appears also to have died. The body of the Jew is thrown over the walls of the city to be left to the scavenger beasts. He revives in time to meet Selim-Calymath on his way to storm the city. He leads the Turks through an underground passage to the city's center where they surprise and defeat the Governor and his men. The Maltese people are put in jail. Selim-Calymath makes Barabas Governor of Malta.

Barabas goes to the former Governor and makes a deal with him that, should he agree to give Barabas great wealth, Barabas will betray Selim-Calymath. The Governor agrees. Barabas sets up a trap in his home whereby Selim-Calymath, sitting in the gallery, will be dropped through a trap door into a boiling caldron. He also wires the great hall where the Turkish soldiers eat with explosives so to destroy the whole Turkish force at once. Barabas instructs the Governor to cut the wire holding the trap door when the sennet sounds (which is also the cue for the detonation of the great hall across the city.)

As Barabas is greeting Selim-Calymath from the gallery, the Governor orders the sennet to sound and cuts the wire so that Barabas falls into the caldron. The Turkish army destroyed, the Governor takes Selim-Calymath prisoner and rids himself of Barabas, who dies slowly in the caldron while cursing one and all.

There are several murders (not counting Nuns, Carpenters, and Turks):

7 by poison (the Machiavel's favorite form),
2 stranglings (including the friar hanged),
2 deaths in a duel,
1 blowing up (of an unspecified number of Turks),
1 boiling in oil (of Barabas).
There is 1 execution (which is really a murder because of Barabas's "setting up" of an innocent friar),
and 0 suicides.

This play's theme is a comparison of POLICY with PROFESSION. POLICY is the sinister underhandedness of the Machiavel (not to be confused with statesmanship). PROFESSION is that which one professes, especially one's religious professions. See especially I.ii.98 and 148-49 and 163 (wherein Barabas says that they profess (make a religion of) policy (sinister political intent)).

The play may be divided into two distinct parts.

The first part encompasses Acts I and II and is the tragic development of Barabas through Abigail's love.
The second part begins with Barabas' loss of Abigail; Acts III, IV, and V degenerate rapidly into farce.

The prologue demonstrates Marlowe's knowledge of the real Machiavelli rather than the biased picture acquired from reading Huguenot (French Protestant) attacks on The Prince.

Shakespeare's Romeo and Juliet (for echoes of nightingale/lark imagery, for balcony scene echoes at II.i.41-42).

Shakespeare's Hamlet (for line echoes at III.iv.68-70; IV.iv.25; V.ii.35).

Shakespeare's King Lear (for one line echo at V.i.21).

Shakespeare's 1 Henry IV (for comparison of Falstaff's rising after presumed dead like Barabas rises in V.i.60—also compare similar acts of the character of Vice in the Morality Plays).

Kyd's The Spanish Tragedy (for lines Marlowe parodies such as at I.ii.268 & 326; IV.i.144; and for action reflected in the evildoer setting up the execution of someone to "take the rap" for his own misdeeds—Lorenzo setting up Pedringano and Barabas setting up the friar Jacomo).