Abstract: In order to further investigate the functional characteristics and potential molecular mechanism of coenzyme Q0 (CoQ0) as an antibiotic adjuvant, the standard strain PAO1 of Pseudomonas aeruginosa was used as the research subject. The cell membrane integrity was evaluated via SYTO^TM9/PI dual-fluorescence staining; the intracellular gentamicin uptake was determined by the inhibition zone method; the intracellular ATP levels were measured by using the chemiluminescence assay, and the intracellular reactive oxygen species (ROS) levels were detected by flow cytometry. The results showed that neither the CoQ0-alone group (600 μg·mL⁻¹ CoQ0) nor the combined treatment group (7.5 μg·mL⁻¹ gentamicin+600 μg·mL⁻¹ CoQ0) disrupted the integrity of bacterial cell membrane. When the concentration of gentamicin was 7.5, 15, 30 μg·mL⁻¹, the intracellular gentamicin uptake in the combined treatment group was significantly lower than that in the gentamicin-alone group. The intracellular ATP levels in both the CoQ0-alone group (600 μg·mL⁻¹ CoQ0）and the combined treatment group (7.5 μg·mL⁻¹ gentamicin+600 μg·mL⁻¹ CoQ0）were extremely significantly lower than those in the blank control group. The intracellular ROS level in the combined treatment group (7.5 μg·mL⁻¹ gentamicin+600 μg·mL⁻¹ CoQ0) was 3.4 times higher than that in the gentamicin-alone group (7.5 μg·mL⁻¹ gentamicin). The above results indicated that the synergistic bactericidal effect of coenzyme Q0 was not achieved by damaging cell membranes or increasing antibiotic uptake, but by inhibiting bacterial energy metabolism, reducing ATP levels, and inducing pronounced oxidative stress, thereby forming a synergistic action of “energy inhibition-oxidative damage” to markedly enhance the bactericidal efficiency of gentamicin.