Aims: To clarify whether an antibacterial surfactant, cetyltrimethylammonium bromide (CTAB), induces superoxide stress in bacteria, we investigated the generation of superoxide and hydrogen peroxide and expression of soxR, soxS and soxRS regulon genes in Escherichia coli cells with the treatment of CTAB. Methods and Results: In situ oxidative stress analyses with BES fluorescent probes revealed that generation of both superoxide and hydrogen peroxide were significantly increased with the CTAB treatment at a sublethal concentration in wild-type strain OW6, compared with the CTAB-resistant strain OW66. The activity of manganese-superoxide dismutase (Mn-SOD), a member of the soxRS regulon proteins, was decreased by the CTAB treatment only in strain OW6. Furthermore, quantitative real-time PCR analyses revealed that expression of the soxRS regulon genes was not upregulated, although soxS was upregulated by the CTAB treatment in strain OW6. Conclusions: Cetyltrimethylammonium bromide treatment led E. coli cells to a generation state of superoxide and hydrogen peroxide. It was also suggested that superoxide generation was caused by inhibiting SoxS function and decreasing Mn-SOD activity. Significance and Impact of the Study: It was revealed that excess superoxide generation in bacterial cells play a key action of antibacterial surfactants.