[Background] Biofilm is a form of bacterial self-protection, which can enhance the resistance of bacteria to antibiotics and host immune response, and causes bacterial resistance and persistent infection. [Objective] To provide a reliable theoretical basis for the development of new natural antibacterial drugs, the inhibition mechanism of carvacrol on the biofilm formation of Staphylococcus aureus was explored. [Methods] Crystal violet staining was used to detect the inhibitory effect of carvacrol on biofilm formation and the clear effect on mature biofilm of tested strains. We used congo red agar to detect the influence of carvacrol on polysaccharide intercellular adhesion (PIA) formation, and used spectrophotometer to detect the influence of carvacrol on extracellular DNA (eDNA) release. RT-PCR analysis was used to determine the effect of carvacrol on the transcription levels of icaA, cidA and sarA. [Results] Carvacrol has strong effects on the inhibition of biofilm formation and the removal of mature biofilm. The inhibition of PIA synthesis and eDNA release by carvacrol at 256 μg/mL was significant. Carvacrol can inhibit the formation of biofilm by inhibiting the transcription of related genes. When carvacrol was 64 μg/mL, the transcription level of sarA decreased by 60.44%±2.91%, cidA by 76.48%±1.67%, and icaA decreased by 70.00%±1.94%. [Conclusion] Carvacrol had significant inhibitory and removal effects on the biofilm of Staphylococcus aureus 25923. Its mechanism is to inhibit the synthesis of PIA and the release of eDNA by reducing the transcription levels of icaA, sarA and cidA genes.