Abstract: The effects of tea tree oil on the biofilm of Methicillin-resistant Staphylococcus aureus (MRSA) and its mechanism of action were studied in this paper . Firstly, the MIC values of tea tree oil against MRSA and 12 clinically isolated strains were determined to be 0.08%~0.32%. It was found that 0.16% of tea tree oil had an inhibitory effect on the formation of MRSA biofilms. Tea tree oil at 0.32% could completely remove mature biofilms of MRSA while 0.16% could significantly reduce the mature biofilms by semi-quantitative crystal violet experiments and CLSM. Preliminary exploration of the biofilm mechanism found that tea tree oil could inhibit the secretion of polysaccharide intercellular adhesion (PIA) and extracellular DNA (eDNA), and the expression level of agr, ica, cid and sar during the formation of MRSA biofilm. Therefore, tea tree essential oil would have a strong anti-biofilm effect, and could inhibite the synthesis of PIA, the secretion of eDNA and the expression of genes related to biofilm formation.