CTBT (7-chlorotetrazolo[5,1-c]benzo[1,2,4]triazine) is an antifungal and chemosensitizing agent that induces oxidative stress in yeast and filamentous fungi and enhances the cytotoxic activity of 5-fluorocytosine and azole antimycotics. This study reports the effect of CTBT on bacterial cells. CTBT inhibited the growth of both Gram-positive and Gram-negative bacterial species. The action of CTBT was bactericidal. In Escherichia coli, CTBT induced an increased formation of reactive oxygen species (ROS), as determined with a ROS specific probe 2′,7′-dichlorodihydrofluorescein diacetate. In zone inhibition assays, bacterial cells were more sensitive to CTBT compared with paraquat, menadione and hydrogen peroxide. The deletion of oxidative stress related genes resulted in increased susceptibility of E. coli mutant strains to CTBT treatment. Exogenous antioxidants such as ascorbic acid, cysteine and glutathione exhibited a protective effect against the growth inhibition induced by CTBT. CTBT may be a useful tool in the studies of ROS generation, oxidant sensing and oxidative stress response in different bacterial species.