Aims This study focuses on the bactericidal mechanism of the new combination of disinfectant glutaraldehyde-didecyldimethylammonium bromide (GA-DDAB) against Escherichia coli. Methods and Results Escherichia coli were exposed to GA-DDAB, and assays for cell morphology, K+ and Ca2+ leakage, H+-ATPase activity and DNA degradation were performed. GA-DDAB damaged the cell wall and disrupted cell-membrane integrity. Leakage of K+ and Ca2+ increased, resulting in significantly lower intracellular concentrations within 60 min of treatment. In addition, H+-ATPase was inactivated and DNA was degraded. Conclusions Leakage of intracellular components indicated that GA-DDAB damaged the cell membrane of E.coli. This may have caused the observed disruption in equilibria of metal ions, inactivation of H+-ATPase, and DNA damage. Significance and Impact of the Study Using a low concentration of GA and DDAB, a new combination disinfectant was developed. GA-DDAB displayed higher antimicrobial activity than treatment with GA or DDAB alone. Therefore, GA-DDAB may be a more cost-effective and efficient antimicrobial agent than others in use today. Furthermore, this study provided a paradigm for developing high-efficiency disinfectant to help address the growing problem of bacterial resistance.