2-Thioxo-3-pyrrolidinecarbaldehyde (TPC), a major product generated from the pungent principle of radish in an aqueous medium was studied to characterize the mode of antimicrobial action. The minimum inhibitory concentration (MIC) of TPC was measured using 25 strains of microbes. The MICs against fungi and bacteria ranged from 50 to 400 μg/ml, while yeasts were more resistant. The effects of TPC on the growth of spores and mycelia of Eurotium chevalieri and on the survival of Staphylococcus epidermidis were investigated, which suggested that the antifungal and antibacterial actions were due to the sporicidal and bactericidal activities. Also, the effects of TPC on uptake of oxygen and radioactive precursors for RNA, DNA, proteins, peptideglycans, and lipids were examined in S. epidermidis. A dose-dependent inhibition of the uptakes of both oxygen and the precursors was observed, suggesting that TPC caused damage to the mitochondrial functions and biosynthetic systems of the biological materials.