She was born in Zell im Wiesental, in Baden-Württemberg, Germany, the daughter of Fridolin Weber. She had three younger sisters (in descending order of age): Aloysia, who was an early love interest of Mozart and sang in his later operas; Constanze, who married Mozart in 1782; and Sophie. Her half-first cousin (son of her father's half brother) was the composer Carl Maria von Weber.
Following her performance as the Queen of the Night at the (highly successful) premiere of The Magic Flute in 1791, she continued to perform this famous role on stage, relinquishing it in 1801 at age 43.
She married twice. Her first husband (married 21 July, 1788 in St. Stephen's Cathedral) was the musician Franz de Paula Hofer (1755–96). Hofer was employed as of 1789 as a violinist at the Imperial court. Her second husband (1797) was the singer Sebastian Mayer (1773–1835). Mayer was the first to perform the role of Pizarro in Beethoven's opera Fidelio.
Josepha Mayer retired from singing in 1805, and died in Vienna December 29, 1819.
Of her singing, the New Grove says, "According to contemporary reports, she commanded a very high tessitura but had a rough edge to her voice and lacked stage presence." The former quality equipped her to take on the very difficult coloratura passages that Mozart wrote into the Queen of the Night's part.