A cationic antibacterial peptide, polymyxin B (PMB), was evaluated as an antifungal antibiotic against various yeasts and filamentous fungi when used in combination with allicin, an allyl sulfur compound from garlic. Allicin was not lethal but could markedly amplify the fungicidal activity of PMB, which was weakly detected with the increase in the plasma membrane permeability in Saccharomyces cerevisiae. Their combined actions caused a dynamic structural damage to the yeast vacuole as judged by the disappearance of its swollen spherical architecture. The vacuole-targeting activity of PMB was similarly amplified in medium with t-butyl hydroperoxide as a substitute for the action of allicin. These findings suggest that the allicin-mediated lipoperoxide production in fungal plasma membrane is the cause of the enhancement in the cellular uptake of PMB as well as its action against the vacuole.