The importance of preventing infectious disease for public health continues to increase, and effective disinfectants are needed to inactive pathogenic microorganisms. Chlorine dioxide (ClO₂) is known as one of the most efficient disinfectants. We studied the inhibitory effect of a novel disinfectant, MA-T, for three species of bacteria (Escherichia coli, Staphylococcus aureus, and Aggregatibacter actinomycetemcomitans). We found that NADH:O₂ oxidoreductase activity (NADH oxidase activity) was markedly decreased in all three species, corresponding to the decrease in colony-forming units following treatment with MA-T. In E. coli, NADH:ubiquinone-1(Q₁) oxidoreductase (NADH-Q₁ dehydrogenase; NDH) activity was decreased following MA-T exposure, indicating that both the NDH-1 and NDH-2 enzymes were targets of this disinfectant. The activity of ubiquinol-1 (Q₁H₂): O₂ oxidoreductase (Q₁H₂ oxidase) also was decreased, indicating that cytochromes bo3 and bd were damaged by MA-T. In S. aureus, NADH-ferricyanide dehydrogenase activity and Q₁H₂ oxidase activity were strongly decreased, suggesting that NDH-2, cytochrome bd, and cytochrome aa3 were targets of MA-T in this species. In A. actinomycetemcomitans, only Q₁H₂ oxidase activity was decreased, indicating that in this species, only cytochrome bd was impaired by MA-T treatment. NADH oxidase activity in membrane vesicles prepared from untreated E. coli was not markedly affected by treatment with MA-T, suggesting that MA-T may attack components of the respiratory chain only in live bacteria (i.e., those possessing a membrane potential), because the membrane vesicles cannot produce the membrane potential.