Salivary histatins are a family of basic histidine-rich proteins in which therapeutic potential as drugs against oral candidiasis is apparent, considering their potent in vitro antifungal activity and lack of toxicity to humans. Histatin 5 (Hst 5) kills the fungal pathogen Candida albicans via a mechanism that involves binding to specific sites on the yeast cell membrane and subsequent release of cellular ATP in the absence of cytolysis. We explored the killing pathway activated by Hst 5 and compared it to those activated by other antifungal agents. The candidacidal activity of human neutrophil defensin 1 (HNP-1) shared very similar features to Hst 5 cytotoxic action with respect to active concentrations and magnitude of induction of nonlytic ATP efflux, depletion of intracellular ATP pools, and inhibitor profile. Hst 5 and HNP-1 are basic proteins of about 3 kDa; however, they have unique primary sequences and solution structures that cannot explain how these two molecules act so similarly on C. albicans to induce cell death. Our finding that HNP-1 prevented Hst 5 binding to the candidal Hst 5 binding protein suggests that the basis for the overlapping actions of these two naturally occurring antimicrobial proteins may involve interactions with shared yeast components.