ABSTRACT Tyrocidines are cationic cyclodecapeptides from Bacillus aneurinolyticus that are characterized by potent antibacterial and antimalarial activities. In this study, we show that various tyrocidines have significant activity against planktonic Candida albicans in the low-micromolar range. These tyrocidines also prevented C. albicans biofilm formation in vitro . Studies with the membrane-impermeable dye propidium iodide showed that the tyrocidines disrupt the membrane integrity of mature C. albicans biofilm cells. This membrane activity correlated with the permeabilization and rapid lysis of model fungal membranes containing phosphatidylcholine and ergosterol (70:30 ratio) induced by the tyrocidines. The tyrocidines exhibited pronounced synergistic biofilm-eradicating activity in combination with two key antifungal drugs, amphotericin B and caspofungin. Using a Caenorhabditis elegans infection model, we found that tyrocidine A potentiated the activity of caspofungin. Therefore, tyrocidines are promising candidates for further research as antifungal drugs and as agents for combinatorial treatment.