The Parisian-born son of a doctor, Berthelot studied medicine at the Collège de France but became interested in chemistry, becoming assistant to Antoine-Jérôme Balard in 1851. He was professor of organic chemistry at the Ecole Supérieure de Pharmacie (1859–76) and professor of chemistry at the Collège de France (1864–1907).

Alcohols were Berthelot's early research interest and he introduced the terms mono-, di-, and polyatomic alcohols. He showed that glycerin was a triatomic alcohol and in 1854 he synthesized fats from glycerin and fatty acids. He carried out a great deal of work on sugars, which he recognized as being both polyhydric alcohols and aldehydes.

Berthelot was one of the pioneers of organic synthesis. Before his time, organic chemists had mainly been concerned with degradations of natural products but Berthelot, in keeping with his logical systematic nature, began with the simplest molecules; his syntheses included methane, methanol, formic acid, ethanol, acetylene, benzene, naphthalene, and anthracene. His favored techniques were reduction using red-hot copper and the silent electric discharge. His methods were somewhat crude and the yields were low. Berthelot's work on organic synthesis was published as Chimie organique fondée sur la synthèse (1860).

Arising from his interest in esterification, Berthelot studied the kinetics of reversible reactions. In 1862, working with Péan de Saint Gilles, he produced an equation for the reaction velocity. This was incorrect but it inspired Cato Guldberg and Peter Waage to enunciate the law of mass action (1864).

In 1864 Berthelot turned to thermochemistry. In his book Mecanique chimique (1879) he introduced the terms ‘endothermic’ and ‘exothermic’ to describe reactions that respectively absorb and release heat. He also introduced the bomb calorimeter for the determination of heats of reaction and investigated the kinetics of explosions.

Berthelot's interest in agricultural chemistry was stimulated by his discovery of nitrogen uptake by plants in the presence of an electrical discharge. In 1883 he established an agricultural station at Meudon, where fundamental work on the nitrogen cycle was carried out. He looked forward to the day when poverty and squalor would be eradicated by the application of synthetic chemistry and new sources of energy.

Berthelot was a pioneer of historical studies in chemistry. In this he was influenced by his friend, the scholar Renan. In later life he became increasingly involved in affairs of state, mostly concerned with education, and in 1895–96 he served as foreign minister.