Abstract: Exploring the exogenous factors that affected the bactericidal efficiency of antibiotics was a key entry point for analyzing the mechanism of bacterial resistance and tolerance, and had important theoretical and practical significance for optimizing the clinical anti-infection treatment. By taking Escherichia coli in the platform period as the research object, the effect of EDTA on the bactericidal activity of gentamicin was evaluated by exogenous addition of ethylenediaminetetraacetic acid (EDTA) combined with gentamicin. The concentration-gradient test and time-dependence test confirmed that: the inhibitory effect of EDTA on the bactericidal activity of gentamicin was significantly concentration-dependent, and this protective effect could persist throughout the whole drug treatment cycle. The results of mechanism exploration revealed that: by reducing the proton motive force (PMF) of Escherichia coli in the plateau phase, reducing the accumulation of intracellular reactive oxygen species (ROS), and alleviating the drug-induced cell membrane damage, EDTA finally weakened the bactericidal efficacy of gentamicin. The further research found that: the protective effect of EDTA was antibiotic class-specific, and only effective against aminoglycoside antibiotics (including gentamicin, tobramycin, streptomycin, kanamycin and amikacin), but had no significant effect on the bactericidal activity of quinolones and β-lactam antibiotics. The study could provide a reference for the clinical evaluation of the rationality of the application of EDTA in the combination of aminoglycoside antibiotics.