The pungent sesquiterpenoid unsaturated dialdehyde, polygodial, exhibited a strong yeastcidal activity against the cells of Saccharomyces cerevisiae, in which production of reactive oxygen species (ROS) at a significant level could be detected with a fluorescent probe. The production of ROS in polygodial-treated cells was further confirmed by its elimination and the accompanying protection against yeastcidal effects in the presence of antioxidants such as L-ascorbate and alpha-tocopherol (alpha-TOH). Polygodial could accelerate ROS production only in cells of the wild-type grande strain but not in those of the respiratory-deficient petite mutant (rho0), indicating the role of the mitochondrial electron transport chain in the production of ROS. Unlike the case with antimycin A which accelerates ROS production by directly targeting the mitochondrial electron flow, polygodial caused depletion of cytoplasmic and mitochondrial glutathione which functions in estiminating ROS inevitably generated during aerobic growth. Polygodial-mediated depletion of intracellular glutathione was possibly dependent on a direct interaction between its enal moiety and the sulfhydryl group of the cysteine in glutathione by a Michael-type reaction.