Count Cagliostro, whose real name is Giuseppe Balsamo, appears in Le Chevalier D'Eon as a part-time peddler and alchemist who works under the mysterious Comte de Saint-Germain. Introduced as a rather aloof and debaucherous man, he is first seen selling perfumes in Russia and has acquired a noticeable taste for fine liquor wherever he travels. He describes his own life as a comedy on stage, expressing a desire to instead create a high class tragedy.

In the story, Cagliostro functions as a rogueish antagonist who conspires with Maximilien Robespierre and his own lover, Lorenza, to trap the protagonist, D'Eon de Beaumont. His primary motivation is to uncover the secret of the Royal Psalms, a supernatural element central to the plot. To achieve this, he seeks to steal the soul that has possessed the French knight, with the intention of using Lorenza as its vessel. Unlike other characters who directly wield the power of the Psalms, Cagliostro himself does not use them; instead, he recognizes their immense power and manipulates Lorenza, who is a user and is not afraid to kill, as his instrument.

Throughout the series, Cagliostro's alliances shift. Initially an ally of Saint-Germain, he later betrays him to work for Maximilien Robespierre. His role places him in opposition to D'Eon and his companions, the Four Musketeers. By the end of the anime, after Robin takes up the name of Maximilien to lead a revolution, Cagliostro is seen standing behind him, indicating his continued presence and influence.