James O'Neil Mayne was born into the prominent Mayne family on 21 January 1861. He was the youngest of five children of Patrick Mayne, a butcher and businessman, and his wife Mary McIntosh. Born and raised in Brisbane, James attended Brisbane Grammar School, earned a Bachelor of Arts at the University of Sydney in 1884, then studied medicine at University College Hospital in London. After obtaining his diploma he returned to Australia to work as a resident medical officer at Brisbane General Hospital (now the Royal Brisbane and Women's Hospital) from 1891 to 1898. In 1898 he was appointed medical superintendent, a position he held until his retirement in 1904. A man of substantial independent means thanks to generational wealth, James financed the purchase of the hospital's first x-ray machine and operating theatre equipment, and donated his salary to the hospital's buildings and grounds committee.

James and his older sister, Mary Emilia Mayne, became renowned philanthropists. Neither married. They were among the first and most significant benefactors of The University of Queensland, donating 280 hectares of farmland at Pinjarra Hills for agricultural education in 1923. In 1926, they contributed £63,000 to acquire over 81 hectares at St Lucia, enabling the University's relocation to its current site.

James died on 31 January 1939 at Moorlands, the family home in Auchenflower, Brisbane. James's estate was valued at £113,334 for probate. The primary assets he shared with his sister, who died a year later, included the Brisbane Arcade, Moorlands, and the Regent building. James and Emilia had identical wills, ensuring their estates would be used to support The University of Queensland Medical School in perpetuity.