Objective: To study the inhibitory ability of Huidu Yinhua Powder, from “Orthodox Manual of External Medicine”, on methicillin?resistant staphylococcus aureus (MRSA) as well as the inhibitory effect of virulence factor α-hemolysin (Hla) activity and biofilm formation, and at the same time, to explore the optimal ratios of Huidu Yinhua Powder, to provide experimental support for the new use of the ancient formula. Methods: The inhibitory effects of Huidu Yinhua Powder and the herbs in the formula on USA300 were analyzed by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and disk diffusion assay (K-B method). Hemolysis assay, neutralization assay, Oligomerization assay, Western Blot (WB) to verify in which form the drug inhibits the activity of the virulence factor alpha-hemolysin (Hla). The biofilm assay was performed to evaluate the inhibitory effect of Huidu Yinhua Powder on biofilm, while finally orthogonal experiments were performed to explore the optimal ratios of Huidu Yinhua Powder. Results: Huidu Yinhua Powder inhibited the MRSA strain with a MIC₉₀ of 64 mg/mL and an MBC of 256 mg/mL, with antibacterial circle diameter of 7.50±0.50 mm. Huidu Yinhua Powder inhibits Hla activity by inhibiting Hla secretion and the minimum effective concentration (MEC) was 16 mg/mL, while the MEC of biofilm was 8 mg/mL. In Huidu Yinhua Powder, Honeysuckle and Astragalus only affected the hemolytic activity of MRSA and the formation of biofilm without inhibiting the growth of bacteria, the hemolytic activity MEC and the biofilm MEC of both of them were 32 mg/mL. Glycyrrhiza had a strong bacterial inhibitory capacity, with a MIC₉₀ of 8 mg/mL and a biofilm MEC of 1 mg/mL, without showing inhibitory hemolytic activity at subinhibitory concentrations. The final orthogonal experiment showed that when the ratio of honeysuckle: astragalus: glycyrrhiza in Huidu yinhua Powder was 1:2:4, the MIC₉₀ was 16 mg/mL, the hemolytic activity MEC was 8 mg/mL, and the biofilm MEC was 4 mg/mL, all of which were the lowest among the nine groups. Conclusion: Huidu Yinhua Powder affects the hemolytic activity and biofilm formation of MRSA at sub inhibitory concentrations, with the optimal ratio of honeysuckle, astragalus, and glycyrrhiza being 1:2:4.