After graduating from the university in his native city of Pisa, Donati joined the staff of the Florence Observatory in 1852, and was appointed director in 1864. He died from bubonic plague in 1873.

Much of his work was concerned with comets. He discovered six new comets, one of which, first appearing in June 1858 has since been known as Donati's comet. He went on in 1864 to make the first observations of a comet's chemical composition. Spectroscopic observation of the 1864 comet produced a line spectrum with three lines named alpha, beta, and gamma by Donati. The three lines were also seen in an 1866 comet by Secchi. The lines were shown by Huggins in 1868 to be due to the presence of carbon.