John Chambers was born in Graceville, Queensland. His father Alfred Henry Chambers was born in Ulster, and his mother Mary, the daughter of John Ferguson, was a Scot. John married Helen Craig in Sydney in 1924. They had two sons.
John Chambers was educated at Tudor House Preparatory School, Moss Vale in NSW and Sydney Grammar School. He attended Melbourne University where he achieved his basic medical degree and later his MD. He studied in England and gained membership of the Royal College of Physicians. He later became a foundation Fellow of The Royal Australasian College of Physicians.
Chambers served in the AAMC in two World Wars as a captain in the First World War and a major in the Second. In between wars he served as honorary physician in both the Alfred and Heidelberg Hospitals. His Second World War experience led him to the 2/4 AGH where he was a valuable and skilful general physician, as he had been before the War at the Alfred Hospital. He was a quiet doctor, though not introverted, and his conversation was predominantly on subjects of importance. In social or professional matters, he was not a man to waste words, though he was acknowledged to be an amusing raconteur.
On 10 April 1941 he was doing a medical round in a military hospital when enemy pilots emerged from the sun and bombed the ward. A bomb dropped almost beside his feet and he died immediately. The military unit lost a first grade physician, as well as a socially valuable member. His death was a great loss to the Heidelberg Hospital and the Alfred Hospital, where prior to his enlistment he was chairman of staff.