Ansilva is waiting-woman to Catalina and Berinthia. Though she should be loyal to both ladies, Ansilva believes that her greatest chance of preferment lies with the elder sister Catalina. Consequently, she assists Catalina's plot to poison Berinthia and have Berinthia kidnapped. She also agrees to add a love potion to Catalina's drink as Count de Montenegro has requested.
ANTONIO DE RIBEIRO
A good friend of Sebastiano, Antonio falls in love with Berinthia, Sebastiano's sister. Catalina, however, has eyes only for Antonio, and her jealousy prompts her to try a combined poisoning and kidnapping scheme to rid her of Berinthia. Antonio foils the scheme when he takes Berinthia to Elvas Castle. Honor-bound to duel for his sister's return, Sebastiano fights and kills Antonio.
BERINTHIA
Berinthia is the vengeful maid. She is the younger daughter of Vilarezo and sister to Catalina and Sebastiano. Duty-bound to reject love offers until her sister is wed, Berinthia nevertheless loves the visiting Antonio, sparking the vile jealousy in her sister who also desires Antonio. Berinthia fails to consume the poison planned for her and is taken to Elvas Castle by Antonio. Here she sees her beloved Antonio and brother Sebastiano fight. Antonio is killed, and Berinthia returned home to Avero. This maid's revenge involves poisoning her sister Catalina and stabbing to death her brother Sebastiano, all before she kills herself in anguish over Antonio's death.
CASTABELLA
Castabella is Antonio's sister. She falls rapidly in love with Sebastiano and is heartsick when he slays Antonio and returns to Avero. Entering Sebastiano's service disguised as a Page, Castabella reveals herself after the deaths of all three of Vilarezo's children. She indicates that her forthcoming life as a nun is likely to be short, and she wishes to be buried near Sebastiano.
CATALINA
The eldest daughter of Vilarezo, Catalina rejects Count de Montenegro as soon as her brother's friend Antonio arrives. Jealous of her sister Berinthia, who is loved by Antonio, Catalina plots to poison her sister and have her kidnapped by Velasco, who loves Berinthia. The poison is never ingested, and Berinthia's captor is Antonio, not Velasco. But the death of Antonio at Sebastiano's hand returns the distraught Berinthia to Avero, and Catalina is poisoned by her vengeful sister.
COUNT DE MONTENEGRO
Montenegro desires Catalina and is fairly certain of her until Antonio arrives and becomes Catalina's sole passion. Montenegro never really understands that Catalina does not love him at all. He battles for her and persuades the waiting-woman Ansilva to put a love potion in Catalina's drink to heighten the passion he thinks the lady entertains for him. He loses all hope in the lady, of course, when Catalina is poisoned by her vengeful sister.
DIEGO
Diego claims his full name is Signior Baltazaro Clere Mautado. As Antonio's servant, Diego carries a secret message from Antonio to Berinthia and overhears Catalina's evil plans for her sister. Diego informs both Berinthia and Antonio of the plot and is instrumental in helping take Berinthia away to supposed safety at Elvas Castle.
DONZEL DEL PHOEBO
A "ghost character." Count de Montenegro tells Catalina that Donzel del Phoebo was a mountebank of valor.
FIRST SERVINGMAN
This Servingman feels Sharkino is an impostor who cannot assist the Third Servingman, James, in locating some missing spoons and napkins.
GASPAR DE VILAREZO
Vilarezo is father to Sebastiano, Catalina, and Berinthia. He insists that Berinthia entertain no love interest until her older sister Catalina is wed. When Berinthia is taken away by Antonio, Vilarezo insists that Sebastiano kill Antonio. The end of the play finds Vilarezo mourning the deaths of all three of his children.
JAMES
James the butler is the Third Servingman and asks for Sharkino's help in locating some missing spoons and napkins.
MAID
This unnamed Maid comes to Sharkino bearing a urinal. She feels she may have lost her maidenhead because she found breeches upon her bed; she is certain that Sharkino can determine the maidenhead's status by examining her "water."
PEDRO
Pedro is the Second Servingman. He introduces James the butler to Sharkino.
ROSICLEERE
A "ghost character." Montenegro mentions Rosicleere to Catalina and describes the gentleman as a "puff."
SCARABEO
Scarabeo is Sharkino's servant. He assists the doctor in his apothecary.
SEBASTIANO
Sebastiano is the son of Vilarezo and brother to Berinthia and Catalina. He brings friend Antonio home to meet his sisters, and both sisters fall in love with Antonio. When Berinthia is taken by Antonio to his castle, Sebastiano must in honor fight his friend, for even though Sebastiano knows Antonio is innocent of wrongdoing, Sebastiano cannot disobey his father's demand to kill Antonio. He slays his friend and brings Berinthia home. He is himself stabbed to death in his bed by the distraught and vengeful maid, Berinthia.
SFORZA
Sforza is an extremely loyal soldier at Elvas Castle, home of Antonio and Castabella.
SHARKINO
Sharkino is a local doctor who runs a sort of apothecary. We find his poisons are all the same despite how he advertises them, and he claims to know by the color of a maid's urine whether she has lost her maidenhead. Sharkino's remedies, if they work at all, are largely placebos.
SIGNIOR BALTAZARO CLERE MAUTADO
Diego claims this to be his full name; he answers to Diego "for brevity's sake."
VELASCO
Velasco desires Berinthia and is sorely upset when she and Antonio seem attracted to one another. His jealousy urges him to take part in Catalina's plot to kidnap Berinthia. Velasco is unaware that Catalina's plan involves his ultimate demise as well. Velasco shows his cowardice at Elvas Castle when he illegally attacks another's opponent. Antonio slays him.
VILLANDRAS
As Antonio's kinsman, Villandras helps Diego and Antonio carry Berinthia away from Avero and to Antonio's Elvas Castle home. Villandras serves as Antonio's second in the challenge from Sebastiano, and he is slain by Sebastiano.