Beatrice-Joanna
Beatrice-Joanna is the daughter of Vermandero. She is betrothed to Alonzo but falls in love with Alsemero. Beatrice-Joanna is selfish and conniving, conspiring with DeFlores to have Alonzo murdered and using her innocent lady-in-waiting, Diaphanta, for her own gain. The play suggests that Beatrice-Joanna, though beautiful on the outside, is no better – perhaps even worse – than DeFlores, who happily murders two people over the course of the play.
Alsemero
Alsemero is Beatrice-Joanna's love interest. He falls in love with Beatrice-Joanna while he is visiting the Spanish town of Alicante, and decides to court her despite her betrothal to another man. Alsemero is obsessed with truth and purity, as evidenced by his potion that he attempts to inflict on Beatrice-Joanna to discern whether she is truly a virgin. At the end of the play, Alsemero is also the one who brings Beatrice-Joanna and DeFlores to justice for their wrongdoings, emphasizing his role as a servant of truth and fairness.
Alonzo
Alonzo is the man to whom Beatrice-Joanna is betrothed at the beginning of the play. Despite Beatrice-Joanna's lack of affection for or interest in Alonzo, he remains excited about their wedding day. After falling in love with Alsemero, Beatrice-Joanna betrays Alonzo and arranges for DeFlores to murder him inconspicuously. In his absence, Alonzo's brother, Tomazo, searches for his brother's killer.
Vermandero
Vermandero is the governor of the castle in Alicante and the father of Beatrice-Joanna. He is the one who betrothed Beatrice-Joanna to Alonzo, and who subsequently engages her to Alsemero after Alonzo is murdered. At the end of the play, Vermandero laments the fact that he did not foresee the wicked nature of his daughter and his servant (DeFlores) sooner, emphasizing his lack of awareness about the scandals in his castle.
DeFlores
DeFlores is Vermandero's servant. He also serves as Beatrice-Joanna's servant and confidant, carrying out her requests in order to gain her affection. He announces early on that he is secretly in love with Beatrice-Joanna, his only aspiration being a sexual relationship with her. At first, Beatrice-Joanna is cruel to DeFlores, harping on his ugly appearance and meek personality. Once he starts doing her bidding, however, she finds herself genuinely attracted to him, even agreeing to have sex with him after he murders Alonzo.
Diaphanta
Diaphanta is Beatrice-Joanna's lady-in-waiting. She is a largely innocent character who, along with Alonzo, becomes one of the casualties of the conspiracy between Beatrice-Joanna and DeFlores. Beatrice-Joanna uses the "maid" potion on Diaphanta first – knowing she is a virgin – to know how to act when Alsemero uses it on her. She also convinces Diaphanta to disguise herself and have sex with Alsemero on the wedding night in order to clear Beatrice-Joanna's name and convince Alsemero that she, too, is a virgin. Tragically, Diaphanta is murdered by DeFlores after he starts a fire in the castle, as DeFlores thinks that Beatrice-Joanna is jealous of Diaphanta.
Jasperino
Jasperino is Alsemero's friend. He does not have a major role in the play other than to flirt crudely with Diaphanta. At the end of the play, it is Jasperino who reveals to Alsemero that something is going on between Beatrice-Joanna and DeFlores.
Tomazo
Tomazo is Alonzo's brother who travels with him to Alicante. Another minor character, Tomazo recognizes right away that Beatrice-Joanna does not want to marry Alonzo. After Alonzo is murdered, Tomazo searches tirelessly for his brother's killer.
Alibius
Alibius is the doctor who runs the madhouse. He is convinced that someone is going to attempt to seduce his (much younger) wife, Isabella, so he instructs his servant to lock her away. Despite his best efforts, however, two nobleman disguised as madmen, as well as Alibius's servant, attempt to force themselves on Isabella. At the end of the play, Isabella pleads with Alibius for more freedom and trust.
Isabella
Isabella is Alibius's younger wife who becomes the victim of a number of unwanted advances while locked away in the madhouse. She is a morally upright and honest woman who fends off the deceptive and unsavory men. At the end of the play, she uses the example of Beatrice-Joanna to request more trust and respect from her husband.
Lollio
Lollio is Alibius's servant and forces himself on Isabella.
Antonio
Antonio is a nobleman from the castle who has disguised himself as a madman to seduce Isabella.
Franciscus
Franciscus is another nobleman who disguises himself as a madman to seduce Isabella.
