Abstract: To further investigate the functional characteristics and potential molecular mechanisms of Coenzyme Q0 as an antibiotic adjuvant, Escherichia coli BW25113 was used as the research object, and four treatment groups were set up: the blank group（Control）, the gentamicin-treated group（Genta）, the Coenzyme Q0-treated group（Q0）, and the combined treatment group（Q0+Genta）. The final concentration of gentamicin in the treated groups was 100 µg·mL⁻¹, and the final concentration of Coenzyme Q0 was 30 µg·mL⁻¹. Meanwhile, hydrogen peroxide（30% solution）was used as the positive control for reactive oxygen species（ROS）determination. Flow cytometry was employed to detect the intracellular ROS level and proton motive force（PMF）level; propidium iodide（PI）staining combined with fluorescence microscopy was used to evaluate cell membrane damage; and scanning electron microscopy was applied to observe cell morphology.The results showed that: Coenzyme Q0 could significantly affect the intracellular ROS level of bacteria, and its combination with antibiotics could generate more ROS, inducing bacteria to enter an oxidative stress state; Coenzyme Q0 could disrupt the normal intracellular energy metabolism of bacteria, thereby reducing their PMF level; outer membrane staining results indicated that the fluorescence intensity of bacteria in the combined treatment group was significantly higher than that in the single-treatment groups, suggesting that Coenzyme Q0 could damage the outer membrane of bacteria and thus increase the uptake of antibiotics; scanning electron microscopy results revealed that the integrity of bacterial membrane morphology in the combined treatment group was disrupted, which further enhanced the bactericidal effect of antibiotics.This study clarified the mechanism by which Coenzyme Q0 enhances antibiotic bactericidal activity by regulating ROS, PMF, and cell membrane structure, providing a theoretical basis for its subsequent development.