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Born in Talloires, France, Berthollet studied medicine at Turin and gained his MD in 1768. He went to Paris in 1772 where he began publishing chemical researches in 1776 and was elected a member of the Académie Française in 1780. His Italian medical degree was not recognized in France so he obtained a Parisian degree in 1778.

When Berthollet published his important paper on chlorine,Mémoire sur l'acide marin déphlogistique (1785), he was the first French chemist to accept Antoine Lavoisier's new system. Unfortunately, he also accepted Lavoisier's erroneous idea that chlorine contains oxygen. In 1784 Berthollet became inspector of a dyeworks and he discovered and developed the use of chlorine as a bleach. He published a standard text on dyeing Eléments de l'art de la teinture (1791).

Berthollet was neither a great manipulator nor a persuasive lecturer, but he did original work in many fields. He analyzed ammonia (1785), prussic acid (hydrogen cyanide, 1787), hydrogen sulfide (1798), and discovered potassium chlorate (1787). Although a convert, he remained skeptical about Lavoisier's oxygen theory of acidity: his analyses showed no oxygen in prussic acid or hydrogen sulfide, despite their undoubted acidity. Berthollet attempted to use his newly discovered potassium chlorate in gunpowder but it proved too unstable, destroying a powder mill at Essones in 1788. More productive were his analyses of iron and steel, which resulted in better quality steel.

After the French Revolution of 1789 Berthollet was a member of various commissions and in 1795 he became a director of the national mint. In 1798 he was entrusted by Napoleon with the organization of scientific work on the expedition to Egypt and he established an Institute of Egypt. On his return to Paris in 1799 Berthollet bought a large house at Arcueil in the suburbs of Paris, where he set up a laboratory and subsequently founded the Société d'Arcueil, which included Pierre de Laplace, Alexander von Humboldt, Jean Biot, Louis Thenard, and Joseph-Louis Gay-Lussac. At Arcueil, Berthollet produced his magnum opus, theEssai de statique chimique (1803), in which he propounded a theory of indefinite proportions. By 1808, following the work of John Dalton, Jöns Berzelius, and Gay-Lussac. indefinite proportions was decisively rejected, but Berthollet's idea that mass influences the course of chemical reactions was eventually vindicated in the law of mass action of Cato Guldberg and Peter Waage (1864).

Berthollet was made a senator in 1804 and in his later years was regarded as the elder statesman of French science.