- Don Pedro, Prince of Aragon: A kind, good Prince who helps Claudio win Hero. It was very common for the superiors of that time to find suitable wives for their men. He later helps Claudio disgrace Hero when he believes that she is unfaithful and he also tricks Benedick and Beatrice to fall in love.
- Benedick, of Padua; companion of Don Pedro: A sarcastic, witty bachelor who swears he will never marry, he later falls in love with Beatrice when he is tricked into believing that she loves him. He is said to be very good in battle and there is hinting at a past relationship with Beatrice, though they do nothing but fight when the story opens.
- Claudio, of Florence; a count, companion of Don Pedro
- Balthasar, attendant on Don Pedro, a singer: Though Don Pedro praises his singing, Benedick calls him a "cat who sounds as if someone is killing it."
- Don John, "the Bastard Prince," brother of Don Pedro and the main villain.
- Borachio and Conrade, followers of Don John: They are the ones who actually initiate the plot to frame Hero as an adulteress. Borachio, who is in a relationship with Margaret, gets her into Hero's clothes and makes love to her on the balcony window, in full sight of Don John, Don Pedro, and Claudio.
- Leonato, governor of Messina: Is Hero's father. He is ready to kill her himself when he believes she has dishonored him, but when he starts to believe her innocence, is ready to turn and kill Claudio instead.
- Hero, Leonato's daughter: Beautiful, sweet, gentle, and demure, she is wrongfully accused of unfaithfulness and publicly humiliated on her wedding day. Wounded by Claudio's anger and her love for him, she swoons, and later pretends to be dead to bring remorse to her beloved. She marries Claudio in the end.
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- Beatrice, niece of Leonato: Hero's witty, older cousin, she has an ongoing 'merry war' of words with Benedick; she mentions once that "I would he had boarded me," insinuating that they had a past relationship. She swears never to marry, but after being tricked into believing that Benedick loves her, falls in love with him. She asks him to avenge Hero's dishonor and he reluctantly agrees to challenge Claudio to a duel.
- Antonio, an old man, brother of Leonato: Offers to fight Claudio after Hero is pronounced "dead."
- Margaret, waiting-gentlewoman attendant on Hero: Borachio's lover, she wears Hero's clothes and is thought to be her mistress. It is not known if she was tricked or was in on the plot.
- Ursula, waiting-gentlewoman attendant on Hero
- Friar Francis, a priest: The priest who believes in Hero's innocence and proposes the plot to pretend that she is dead.
- Dogberry, the constable in charge of Messina's night watch: An idiot with a too-large sense of self-importance, he continuously botches everything he tries to do but is indirectly responsible for Hero's public redemption from disgrace.
- Verges, the Headborough, Dogberry’s partner
- A Sexton, the judge of the trial of Borachio
- The Watch, watchmen of Messina
- A Boy, serving Benedick
- Attendants and messengers
- Innogen, a ghost character included in early editions as Leonato's wife
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