- The Duchess – The protagonist, sister to Ferdinand and the Cardinal. At the beginning she is a widow whose brothers take every precaution to keep from marriage, though later she secretly marries Antonio, and for this her brothers arrange to have her strangled. She is described as having a sweet countenance and noble virtue, unlike her brothers. She is also witty and clever, helping her keep up with her brothers' banter, and has a tenderness and warmth which they lack. She has three children, two sons and a daughter by Antonio. (There is an inconsistency surrounding earlier children by her deceased husband, put down to a careless mistake by Webster.) Based on Giovanna d'Aragona, Duchess of Amalfi
- Antonio Bologna – Antonio returned from France, full of scorn for the Italian courtiers whom he sees as more corrupt than the French. Antonio is the steward of the Duchess of Malfi's palace. His honesty and good judgment of character are traits well known to the other characters. He accepts the Duchess' proposal of marriage because of her disposition rather than her beauty. Her marrying beneath her status is a problem, however, and their marriage has to remain a secret, as Antonio shares neither her title nor her money.
- Delio – A courtier, who tries to woo Julia. Based on Matteo Bandello's self-depiction under this name, his purpose is to be the sounding board for his friend Antonio. Because he asks so many pertinent questions, he serves as a source of important information to the audience, and is privy to the secrets of Antonio's marriage and children.
- Daniel de Bosola – A former servant of the Cardinal, now returned from a sentence in the galleys for murder. Publicly rejected by his previous employer the Cardinal, he is sent by Ferdinand to spy on the Duchess as her Provisor of Horse.[Note 1] (Ferdinand hopes to keep her away from marriage.) Bosola is involved in the murder of the Duchess, her children, Cariola, Antonio, the Cardinal, Ferdinand, and a servant. Witnessing the nobility of the Duchess and Antonio facing their deaths, he finally feels guilty, and seeks to avenge them. This change of heart makes him the play's most complex character. A malcontent and cynic, he makes numerous critical comments on the nature of Renaissance society. (He is based on the historical Daniele de Bozolo, about whom little is known.)
- The Cardinal – The brother to the Duchess and Ferdinand. A corrupt, icy cardinal of the Roman Catholic Church who keeps a mistress. He has arranged a spy (Bosola) to spy upon his sister – all this on the quiet, however, leaving others ignorant of his plotting. Of remorse, love, loyalty, or even greed, he knows nothing, and his reasons for hating his sister are a mystery. (Historically, his name was Luigi d'Aragona.)
- Ferdinand – The Duke of Calabria and twin brother of the Duchess. Unlike his rational brother the Cardinal, Ferdinand has rages and violent outbursts disproportionate to the perceived offence. As a result of his regret for hiring Bosola to kill the Duchess, he gradually loses his sanity—he believes he is a wolf and digs up graves (lycanthropia). (In reality, his name was Carlo, Marquis of Gerace.)
- Castruchio (Castruccio) – An old lord. His name plays on the word "castrated", suggesting impotence. He's the conventional elderly man with a young, unfaithful wife (Julia). He is genial and easygoing, attempting to stay on good terms with all.
- Roderigo – A courtier
- Grisolan – A courtier
- Silvio – A courtier
- Pescara – A marquis, possibly Fernando d'Avalos
- Cariola – The Duchess's waiting-woman who is privy to her secrets. She witnesses the Duchess's wedding and delivers her children. She dies tragically by strangling following the murder of the Duchess and the youngest children. Her name plays on the Italian carriolo, meaning "trundle-bed", where personal servants would have slept.
- Julia – Castruchio's wife and the Cardinal's mistress. She dies at the Cardinal's hands from a poisoned Bible.
- Malateste – A hanger-on at the Cardinal's court. The name means 'headache'. Referred to as a "mere stick of sugar candy" by the Duchess, he is yet another interchangeable courtier serving the sycophantic court.
- Doctor – Sent for to diagnose Ferdinand's madness and his supposed "lycanthropia".
There are also minor roles including courtiers, servants, officers, a mistress, the Duchess's children, executioners, etc.
