The active ingredient in Dolethal is pentobarbital, which is a barbiturate. Barbiturates act by depressing the central nervous system, inhibiting accumulation of calcium in nervous tissue, which leads to liberation of norepinephrine, acetylcholine, glutamate and gamma-aminobutyric acid. This leads to sedation and coma.

Barbiturates also act on cardiac and respiratory systems bringing on apnoea and cardiac arrest.