Abstract: To prepare a postbiotic with good antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), and to study its antibacterial effect and action mode, a postbiotic named YDFF was prepared by co-fermenting Lactococcus lactis subsp Postbio-F3, Lactobacillus paracasei Postbio-P6 and Lactobacillus fermentans Postbio-Q7, its antibacterial activity against MRSA was determined by both inhibition zone and growth curve, and the active ingredients exerting antibacterial activity were preliminarily identified. The antibacterial mechanism of YDFF on MRSA was investigated by detecting the effects of YDFF on MRSA biofilm formation, ultrastructural changes, DNA leakage, and ROS generation. Furthermore, the effect of YDFF on the antibiotic resistance of MRSA to methicillin was explored by chessboard method. The results showed that the postbiotic YDFF had good antibacterial activity against MRSA (The diameter of inhibition zone was 19.1±1.5 mm) with wide pH tolerance range (pH3.0~9.0). The extracellular polysaccharides extracted from YDFF had no antibacterial activity, but protease K could completely inactivate its antibacterial activity, indicating that the main antibacterial active substance in YDFF was protein. It was demonstrated that YDFF exhibited antibacterial activity by several ways including inhibiting the formation of biofilms, destroying the cell membrane integrity which resulted in the leakage of DNA, and increasing the concentration of intracellular ROS. Additionally, when YDFF was used combined with methicillin, the fractional inhibitory concentration index (FICI) was significantly decreased to 0.38, indicating YDFF could reduce the antibiotic resistance of MRSA to methicillin. Taken together, YDFF exhibited good antibacterial activity on MRSA by inhibiting the biofilm formation, destroying membrane structure and increasing the intracellular ROS, and reduced the antibiotic resistance to methicillin, which would provide a theoretical basis for the use of postbiotics in food and medicine.