Harnoncourt was born into a noble family who were related to the Holy Roman Emperors and other European royalties. His full name was Count Nikolaus de la Fontaine und d'Harnoncourt-Unverzagt. He was brought up in Graz, Austria and studied music in Vienna.
From 1952 to 1969 he was a cellist with the Vienna Symphony Orchestra. He soon became interested in performing old music with period instruments. He started a group called Concentus Musicus Wien. They tried to make the music of composers from the Baroque period sound like it had done when it was written. Harnoncourt played the viola da gamba as well as the cello. In 1970 he conducted Monteverdi's opera Ritorno d'Ulisse in Patria at La Scala, Milan.
In 1971, Harnoncourt started to work with the conductor Gustav Leonhardt. They recorded all of J.S. Bach's cantatas. This project took until 1990 to finish. He also recorded Bach's St. Matthew Passion. He performed with many other orchestras, including the Royal Concertgebouw Orchestra. Often they were orchestras who used modern instruments, but he tried to make them play with an understanding of a historical style (bowing, phrasing etc). Like other musicians working with historical instruments, he gradually realized that music from later periods such as the Classical music period and even the Romantic period used to sound different from what we usually hear today. He recorded the symphonies and concertos of Beethoven. He received many awards for these recordings.
Harnoncourt and his wife Alice have had four children.