The play's central theme is revenge, woven throughout the plot with varying degrees of moral justification. This theme is introduced at the outset when Andrea's ghost seeks retribution against Balthazar for killing him in battle, even though the ghost cannot directly enact this revenge. The next character motivated by revenge is Bel-Imperia, who also targets Balthazar because he killed her lover, Andrea. Initially, she plans to use Horatio as her tool for vengeance, and when Horatio is murdered, her desire for revenge intensifies.

The third character seeking revenge is Balthazar, who aims to retaliate against Horatio for capturing him in battle and hindering his pursuit of Bel-Imperia.

The final and most significant example of the revenge theme is Hieronimo, who seeks justice for the murder of his son, Horatio. Hieronimo’s wife, Isabella, shares his thirst for vengeance.

Although Horatio’s murder occurs later in the second act, Hieronimo’s quest for revenge becomes the play’s main focus, as he has suffered the greatest injustice. One could argue that Andrea has little reason to seek revenge on Balthazar since their encounter was a fair battle. However, Hieronimo has a clearly justifiable cause. Additionally, the audience witnesses Horatio’s murder directly, while Andrea’s death is merely recounted, adding more emotional weight to Hieronimo’s situation.

Once the revenge plot is established, the question of how it will be carried out and by whom arises. Hieronimo initially considers taking matters into his own hands. However, Isabella suggests that “the heavens are just” and that time will expose the villains and bring them to justice.

Shortly after, in act 3, scene 2, Hieronimo, frustrated by his ignorance of the murderer’s identity, begins to question the idea of cosmic justice. In lines 9–11, he directly appeals to the “sacred heavens,” questioning the fairness of their actions:

Shall unrevealed and unrevenged pass,
How should we term your dealings to be just,
If you unjustly deal with those that in your justice trust?

Immediately after this plea, Hieronimo discovers a letter from Bel-Imperia, revealing that the murderers are Balthazar and Lorenzo, suggesting that cosmic justice may indeed be responding to his situation. Nevertheless, Hieronimo begins to believe he must exact the revenge himself. He is deeply concerned with the notion of justice and refuses to act until he is sure of the murderers’ guilt. When Hieronimo finally obtains undeniable proof of their identity, he thanks heaven, believing that the gods have ensured the murder will not go unpunished.

Concerned with achieving justice, Hieronimo decides to bring his case before the king, seeking secular justice. However, Lorenzo steps in and stops him from disclosing the entire truth to the king. When this attempt fails, and after briefly contemplating the Christian belief that vengeance is God's domain, Hieronimo decides to take action himself. He rationalizes his decision by believing that his actions are "wrought by the heavens." Most modern readers feel Hieronimo goes too far, particularly because he also kills the Duke of Castile, an innocent man who becomes a target solely because he is Lorenzo’s father.

Hieronimo's extreme measures make it hard to argue that he is merely a tool of divine justice. It appears he has moved beyond being a righteous avenger to becoming a murderer and villain. His final actions suggest that any human attempt to deliver justice is risky and prone to mistakes. A clear example of the unreliability of human justice is demonstrated in the trial and execution of Pedringano. Although Pedringano may deserve his punishment, the legal proceedings fail to prove he acted under Lorenzo's orders, allowing Lorenzo to evade justice in this instance.