Coumarin derivatives as antifungal bioactive agents have been considered for many years. However, their mechanism of action is not very clear. In the current paper, antifungal activity of natural and synthetic coumarins are studied against Cryptococcus neoformans and Saccharomyces cerevisiae. Mechanism of pH-dependent activity of coumarin compounds was investigated using 31P-NMR spectra and even intracellular estimate of the pH values. The results showed that coumarin derivatives have inhibitory effects without any noticeable side effects through a selective ATPase inhibitory mechanism.