Born Ada May Campbell, her father died when she was 13 years old and her stage-minded mother, in need of money, encouraged May and her younger sister Flora to perform. Creating a singing act, the young girls debuted in nearby Buffalo, New York in December of 1874. By the fall of 1877, their career had progressed to where they were booked to appear at New York's Metropolitan Theater then at the Tony Pastor Theatre, a popular New York City music hall.

The Irwin sisters proved popular enough to earn regular spots for the ensuing six years after which a 21-year-old May Irwin set out on her own. She joined Augustin Daly's stock company where she made her first appearance on the theatrical stage. An immediate success she went on to make her London stage debut at Toole's Theatre in August of 1884. In 1886 her husband of eight years, Frederick W. Keller, died unexpectedly.

By the early 1890s, May Irwin had married a second time and developed her career into that of a leading vaudeville performer with an act known at the time as "Coon Shouting" in which she performed African American influenced songs. In the 1895 Broadway show The Widow Jones, she introduced "The Bully Song" which became her signature number. The performance also featured a lingering kiss which was seen by Thomas Edison who hired Irwin and her co-star John Rice to repeat the scene on film. In 1896, the Kinetoscope production, The Kiss, became the first screen kiss in cinematic history.

In addition to her performing and singing, May Irwin also wrote the lyrics to several songs, including "Hot Tamale Alley," with music written by George M. Cohan. In 1907 she married her manager, Kurt Eisfeldt, the same year she began making records for Berliner/Victor.

May Irwin's buxom figure was much in vogue at the time and combined with her charming personality, for more than thirty years she was one of America's most beloved performers. In 1914, she made her second silent film appearance, this time in the feature-length adaptation of George V. Hobart's play, Mrs. Black is Back.

A highly paid performer, Irwin was a shrewd investor and became a very wealthy woman. She spent a great deal of time at a summer home on secluded Club Island in the Ontario part of the Thousand Islands and at her winter home on Merritt Island, Florida before retiring to a farm near Clayton, New York where a street would eventually be named in her honor.