Theiler was born in Pretoria, then the capital of the South African Republic (now South Africa); his father Arnold Theiler was a veterinary bacteriologist. He attended Pretoria Boys High School, Rhodes University College, and University of Cape Town Medical School, graduating in 1918. He left South Africa for London to study at St Thomas's Hospital Medical School, King's College London, and at the London School of Hygiene and Tropical Medicine. In 1922, he was awarded a diploma in tropical Medicine and hygiene; he became a licentiate of the Royal College of Physicians of London and a member of the Royal College of Surgeons of England.