By far the best-known achievements in the chapel are the work of Michelangelo. Across the ceiling he painted nine episodes from Genesis. There are representations of the stages of creation, Adam and Eve's temptation and fall, and Noah and the Deluge.

Frescoes by Perugino, Pinturicchio, Botticelli, Ghirlandaio, Rosselli, and Signorelli cover the side walls of the chapel. They depict scenes from the lives of Moses and Jesus, symbolizing the reign of law and of grace, respectively.

In 1980 cleaning and restoration of Michelangelo's frescoes began with the test cleaning of small areas; full-scale work started the following year and was completed in 1994.

The cleaning became controversial as it proceeded, as many experts accused the restorers of distorting or destroying the frescoes, but others defended the work, astonished at the colorist that the restoration revealed the artist to be. The cleaning of chapel frescoes by Botticelli, Perugino, and other painters was completed in 1999.

More than twenty years after completing his ceiling masterpiece, Michelangelo also painted the Last Judgment on the wall behind the altar.