- Monticelso – A Cardinal, later Pope Paul IV.
- Francisco De Medici – Duke of Florence; in Act V disguised as the Moor, Mulinassar.
- Brachiano – Otherwise Paulo Giordano Orsini, The Duke of Brachiano, husband of Isabella, and in love with Vittoria.
- Giovanni – Brachiano's son by Isabella.
- Lodovico – Sometimes Lodowick, an Italian Count in love with Isabella.
- Antonelli – Ludovico's friend and conspirator.
- Gasparo – Ludovico's friend and conspirator.
- Camillo – Vittoria's husband, nephew of Monticelso.
- Carlo – Attendant of Brachiano, in league with Francisco.
- Pedro – Attendant of Brachiano, in league with Francisco.
- Hortensio – One of Brachiano's officers.
- Marcello – An attendant to the Duke of Florence; Vittoria's younger brother.
- Flamineo – Vittoria's brother. Brachiano's secretary.
- Isabella – Francisco De Medici's sister; first wife of Brachiano
- Vittoria Corombona – a Venetian lady, sister of Flamineo. first married to Camillo – afterwards to Brachiano
- Cornelia – Mother to Vittoria, Flamineo, and Marcello
- Zanche – Moor servant to Vittoria; in love with Flamineo, then Francisco
- Ambassadors, Courtiers, Lawyers, Officers, Physicians, Conjurer, Armourer, Attendants, Matron of the House of Convertites, Ladies.
- Doctor Julio.
- Christophero, One of the line-less "ghost" characters who helps Doctor Julio murder Isabella.
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