Highlights • We examined the antifungal activity of 3-bromopyruvic acid (3-BP). • 3-BP could be a novel antifungal agent against Cryptococcus neoformans. • 3-BP acts on C. neoformans through fast depletion of intracellular ATP. • Its different uptake and accumulation explain different susceptibility of fungi. • Susceptibility is also related to a natural level of intracellular glutathione.