The present study aimed to investigate the antibacterial effect and mechanism of a carvacrol-rich Oregano essential oil (OEO) on the inactivation of Staphylococcus aureus. A label-free proteomic analysis was applied to analyze the regulatory networks of S. aureus in response to 1/2 MIC OEO-treatment stress. These findings deepened our understanding regarding S. aureus survival and metabolism responses to the OEO treatment and suggested that the carvacrol-rich OEO could be used in food production environments to effectively control S. aureus.