Little is known about Isaac's early life, but it is probable that he was born in Flanders. It is known that he was writing music by the mid 1470s, and the first documentary reference to him is from 1484, when he was court composer at Innsbruck. The following year, he entered the service of Lorenzo de Medici at Florence, where he was organistist, choir master, and teacher to Lorenzo's children. By 1497, Isaac was in the employ of Emperor Maximilian I. He travelled widely in Germany, and is credited with having a big influence on German composers of the time. Isaac returned to Florence in 1514, and died there in 1517.
Isaac composed a wide variety of music, including masseses, motets, German and Italian songs and instrumental music. He was one of the most prolific composers of his time, but his work has been largely neglected in favour of Josquin (although the composer Anton Webern wrote his thesis on Isaac). His best known work is probably the lied Innsbruck, ich muss dich lassen, of which he made at least two versions. It is possible, however, that the melody itself is not by Isaac, and only the setting is original. The same melody was later used as the theme for the Lutheran chorale O Welt, ich muss dich lassen, which was the basis of works by Johann Sebastian Bach and Johannes Brahms.
On his death, Isaac left his Choralis Constantinus incomplete. It is the first known complete setting of the Proper of the Mass for the entire year, containing around one hundred settings. Isaac's student Ludwig Senfl completed the set, but it was not published until 1555, after his death. Isaac also wrote nearly 40 Mass Ordinaries.