Mackerras was born in Schenectady, New York in the United States of America to Australian parents, and moved with them to Sydney at an early age. He studied at the conservatory there and eventually became principal oboist of the Sydney Symphony Orchestra. He settled in England in 1946 and went to Prague to study conducting with Václav Talich. Returning to England, he worked at Sadler's Wells Opera (now English National Opera) and then was principal conductor of the BBC Concert Orchestra from 1954 to 1956. He directed the Hamburg State Opera from 1965 to 1969, English National Opera from 1970 to 1978 and Welsh National Opera from 1987 to 1992. He was knighted in 1979.
Mackerras has performed a wide range of repertoire, but is particularly closely associated with the operas of Leos Janacek - in 1951 he conducted the British premiere of Katya Kabanova, which had been written thirty years earlier. He is also well known for his knowledge of Mozart's operas, which is encyclopaedic (it has been said that if the scores of Mozart's operas were all somehow destroyed, Mackerras would be able to reconstruct them entirely from memory). He has often been careful to give historically authentic performances of these works: in a 1965 performance of The Marriage of Figaro he ensured that ornamentation was added in a way that Mozart would have recognised.
Mackerras has also arranged music by Arthur Sullivan for the ballet Pineapple Poll, and music by Giuseppe Verdi for the ballet The Lady and the Fool.