- The main character, Isabella, is a very virtuous and chaste young woman who faces a difficult decision when her brother is sentenced to death for fornication (unlawful sex). Isabella does not approve of her brother's actions at all, but she pleads for his life out of loyalty and sisterly devotion. Isabella is a spiritual person who starts off wanting to become a nun.
- The other central figure is the Duke, who spends most of his time dressed as a friar in order to observe what is happening in his absence. The Duke is unfailingly virtuous, good, and kind-hearted. He tends to rule a little softly, which is why he enlists Angelo's help.
- Isabella's brother Claudio is a young man sentenced to death for impregnating an unmarried woman. He was engaged to her by a common-law agreement, but they had sexual intercourse before the legal marriage took place. Claudio depends less on the guidance of laws and religious practices than on his sister.
- Angelo is the villain of the play, a man who rules strictly and without mercy. He has his own weaknesses, however, and he is loathsome more for his hypocrisy than for anything else. He presents Isabella with a difficult proposition and then does not even hold up his end of the bargain.
- Escalus is a wise lord who advises Angelo to be more merciful. He is loyal to the Duke and seeks to carry out his orders justly, but cannot go against Angelo's will.
- Lucio, described by Shakespeare as a "fantastic," is a flamboyant bachelor who provides much of the play's comedic content. He is a friend of Claudio's and tries to help him.
- Mariana was supposed to marry Angelo, but he called the wedding off when she lost her dowry in a shipwreck that killed her brother.
- Mistress Overdone runs a brothel in Vienna.
- Pompey is a clown who also works for Mistress Overdone.
- The provost runs the prison and is responsible for carrying out all of Angelo's orders.
- Elbow is a dim-witted constable who arrests people for misconduct, particularly of the sexual variety. He speaks in malapropisms and provides comic-relief throughout the play.
- A long-term prisoner in the jail, Barnadine is sentenced to be executed together with Claudio. The Duke originally considers him hopeless and therefore dispensable but later changes his mind.
- Claudio's lover, she is pregnant with his baby.