Abstract: Identifying exogenous factors that influence antibiotic bactericidal efficacy is a key entry point for understanding the mechanisms underlying bacterial resistance and tolerance, and holds significant theoretical and practical value for optimizing clinical anti-infective treatment strategies. Using stationary-phase Escherichia coli as the model organism, this study evaluated the effect of ethylenediaminetetraacetic acid（EDTA）on the bactericidal activity of gentamicin by treating cells with gentamicin alone or in combination with EDTA. Concentration-gradient and time-dependent assays demonstrated that the inhibitory effect of EDTA on gentamicin-mediated killing was strongly concentration dependent and persisted throughout the entire treatment period. Mechanistic investigations revealed that EDTA reduced the proton motive force（PMF）, decreased intracellular reactive oxygen species（ROS）accumulation, and alleviated antibiotic-induced cell membrane damage in stationary-phase E. coli, ultimately weakening the bactericidal efficacy of gentamicin. Further analyses showed that the protective effect of EDTA was antibiotic-class specific: it was observed only with aminoglycosides—including gentamicin, tobramycin, streptomycin, kanamycin, and amikacin—and had no significant impact on the killing activities of quinolone or β-lactam antibiotics. Together, these findings provide important experimental evidence for re-evaluating the clinical rationality of combining EDTA with aminoglycoside antibiotics in therapeutic applications.