AimsQuaternary ammonium compounds (QACs), including benzalkonium chloride (BAC) and cetylpyridinium chloride (CPC) are cationic surfactants and have been used widely as general disinfectants in the medical field due to their strong antibacterial effects and low cytotoxicity to human cells. 4,4-(,-hexametylenedithio) bis (1-octylpyridinium bromide) (4DTBP-6,8) is one of the potent bis-QACs synthesized to improve the antimicrobial activities ofmono-QACs such as BAC. This study aimed to assess the effectiveness of 4DTBP-6,8 against Pseudomonas aeruginosa, a prevalent hospital pathogen. Methods and ResultsThe minimum inhibitory concentrations of 4DTBP-6,8, CPC and BAC against P. aeruginosa were measured. 4DTBP-6,8 exhibited strong antibacterial activity. We assessed the bactericidal effects of QACs against P. aeruginosa under certain conditions and their cytotoxicities in human epithelial cells using lactate dehydrogenase (LDH) release. 4DTBP-6,8 exerted excellent bactericidal effects against high concentrations of bacteria, biofilm cells and even in the presence of contaminated proteins. Cellular LDH was not released by the treatment with 4DTBP-6,8. Conclusions4DTBP-6,8 exhibited the strongest bactericidal activity against P.aeruginosa among the three QACs tested without any cytotoxicity. Significance and Impact of the StudyThe potent bis-QAC, 4DTBP-6,8 has the potential to be an effective disinfectant in preventing hospital infections caused by P. aeruginosa.