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Coenzyme Q0 (CoQ0), one of benzoquinone compounds, has been demonstrated to possess antineoplastic, anti-inflammatory and antioxidant activities. However, its antimicrobial effect has not been extensively reported. In this study, antimicrobial activity of CoQ0 against Staphylococcus aureus was evaluated by measurement of inhibition zone, minimum inhibitory concentration (MIC), and growth curves. Time-kill assay was performed to assess the bactericidal activity of CoQ0 against S. aureus in tryptone soya broth and pasteurized milk. The possible mechanism of action was explored through measuring changes in intracellular ATP concentrations, membrane potential, and cell morphology. Furthermore, propidium iodide (PI) staining assay was performed to evaluate the effect of CoQ0 on cell membrane integrity. The MIC of CoQ0 against tested strains ranged from 7.8 to 62.5 μg/mL. CoQ0 at 2 × MIC showed bactericidal effect on S. aureus in tryptic soy broth (TSB) and pasteurized milk. Decrease in intracellular ATP concentration and membrane potential were detected when cells were treated with CoQ0. PI staining demonstrated destruction of bacterial cell membrane. CoQ0 also induced abnormal cell morphological changes, as confirmed by field emission scanning electron microscopy. These findings suggested that CoQ0 exhibited antimicrobial effect on S. aureus , which was partly because of its ability to damage cell membrane.