Staphylococcus aureus (S. aureus) is a typical kind of symbiotic bacteria, which can cause human pneumonia, food poisoning, and other health problems. Nowadays, the corresponding prevention and treatment have been a hot issue of general concern in related research areas. However, the mechanism of action against S. aureus is not well understood. In order to tackle such problem, we used broth microdilution to discuss the antibacterial effect of 5-methyl-2-isopropylphenol and determine inhibitory concentration. In addition, membrane potential and lipid peroxidation levels were also measured under experimental conditions. The experimental results suggested that 300 μg/mL thymol might cause cell membrane damage and decrease of NADPH concentration and increase of NADP+ and lipid peroxidation level. In such condition, thymol has the potential to result in membrane rupture and disruption of cellular homeostasis. Furthermore, we also found that NOX2 is involved in maintaining the balance of NADPH/NADP+ in cells. Finally, our work confirms that NOX2 is a potential downstream target for thymol in the cell. Such target can provide specific guidance and recommendations for its application in antifungal activity. Meanwhile, our study also provides a new inspiration for the molecular mechanism of thymol's bacteriostatic action.