Outside the Eisenstein residence, Alfred serenades his former lover, Rosalinde, who is now married to Gabriel von Eisenstein. Rosalinde’s housemaid, Adele, wishes she could attend the glamorous party that Prince Orlofsky is throwing. When Rosalinde refuses to give her the night off, Adele pretends her aunt is sick. Alfred continues to profess his love, and although Rosalinde initially resists, she eventually agrees to a rendezvous later.

Eisenstein returns home with his lawyer, Dr. Blind, after having just been sentenced to eight days in jail for hitting a police officer. His friend Falke urges him to delay jail for a night so they can both attend Orlofsky’s party. Unbeknownst to Eisenstein, Falke also invites Rosalinde, offering her the chance to catch her husband flirting with other women. When Eisenstein departs for “prison,” Rosalinde bids him farewell.

Alfred returns for a romantic nightcap, but they are interrupted by the prison warden Frank, who has come to arrest Eisenstein. To protect her reputation, Rosalinde convinces Alfred to pose as her husband and leave with Frank.

At Prince Orlofsky’s extravagant party, guests gossip about their enigmatic host. Orlofsky is skeptical when Falke promises him an entertaining evening, but he agrees to see where the night takes them.  

Adele shows up and surprises her sister Sally, who is a professional dancer, by posing as a mysterious starlet named Tanya. Eisenstein is masquerading as a Frenchman. He recognizes Adele but she manages to preserve her disguise. One of the other partygoers is prison warden Frank, who pretends to be a producer to impress Sally and Adele.  

When Rosalinde makes her entrance in a mask and costume, she is incensed to see her husband indeed flirting with other women. She slyly steals his favorite pocket watch after capturing his attention. As midnight approaches, Falke recounts the story of his infamous nickname, Dr. Fledermaus (“Dr. Bat”), which he received courtesy of a past prank by Eisenstein. The crowd toasts to champagne and brotherhood as the revelry continues late into the early hours of the morning, when Eisenstein must rush off to jail. 

At the jail, the grumpy warden Frosch is annoyed by Alfred’s non-stop singing. Frank arrives, slightly inebriated and reminiscing about the previous night’s escapades. Adele and Sally come to the prison hoping that Frank can help them advance their stage careers.  

When Eisenstein arrives at the jail, he is surprised to discover his cell is already occupied by a man who was found with his wife. Eisenstein disguises himself as Blind in order to confront the imposter. Rosalinde rushes in, seeking Alfred’s release. When Eisenstein reveals himself to Rosalinde, she defiantly produces his watch as proof of his own would-be infidelity. The scene dissolves into a brawl, and Orlofsky arrives to break it up. As peace is restored, the entire party sings a final toast to the pleasures of champagne and love.