The mechanism of polygodial antifungal action was studied using Saccharomyces cerevisiae. It inhibited the incorporation of radioactive precursors into macromolecules in whole cells. However, no specificity in the inhibition of precursor incorporation was observed among various species of macromolecules, i.e., DNA, RNA, protein, and polysaccharide. It also inhibited the exogenous but not endogenous respiration of the cells. Polygodial greatly enhanced the leakage of cellular constituents from the treated cells, and the examination under an electron microscope revealed structural damage of the cell membranes of treated cells. These results support the conclusion that polygodial acts primarily by damaging the permeability barrier of the yeast cells.