Objective To explore the antimicrobial activity of antiparasitic drug dichlorophenol (DIC)against Staphylococcus aureus (S. aureus). Methods Antimicrobial susceptibility and resistance inducing ability of S. aureus against DIC was detected by micro-broth dilution assay and disc diffusion test; bactericidal efficacy of DIC was assessed by time-killing curve; inhibitory effect of DIC on the formation of biofilm and eradicating of formed biofilm of S. aureus was detected by crystal violet and XTT staining; cytotoxicity of DIC was detected by cell counting kit-8 (CCK-8), and a mouse model of skin abscess infection was constructed to detect the in vivo antimicrobial activity and toxicity of DIC. Results The minimal inhibitory (MIC) and minimal bactericidal concentration (MBC) of DIC against S. aureus standard strains were 2-4 μg/mL and 2-8 μg/mL, respectively. The MIC and MBC of DIC against S. aureus clinical strains were 2-8 μg/mL and 8-32 μg/mL, respectively. Disc diffusion test indicated the obvious concentration-dependent bacterial growth inhibitory effects of DIC on S. aureus standard strains. Time-killing assay revealed that DIC concentration of 4×MIC was found to reduce the viable bacterial cells of S. aureus standard strains ATCC 29213 and ATCC 43300 from (5.51±0.27) Log₁₀ CFU/mL and (5.44±0.08) Log₁₀ CFU/mL to the limit of detection after 2 hours and 4 hours treatment, respectively. No drug-resistant mutant strains of S. aureus were found after 15 consecutive passage of DIC with subinhibitory concentration on bacteria. 2 μg/mL DIC could significantly inhibit the formation of S. aureus biofilm and reduce the total amount of biofilm from (100±7.49)% to (11.12±2.86)% (P<0.001). 2 μg/mL DIC significantly eradicated the formed S. aureus biofilm and reduced the total biofilm from (100±10.34)% to (42.53±16.87)% (P<0.001). DIC could significantly reduce the viable bacterial load of S. aureus in abscess in mice, and reduce the number of viable bacteria from (9.54±0.46) Log₁₀ CFU/abscess to (7.78±0.62) Log₁₀ CFU/abscess (P<0.05). Hematoxylin-eosin staining result showed that DIC could significantly reduce the abscess area and inflammatory cell infiltration in mice tissue, and was well tolerated in vivo. Conclusion DIC has low cytotoxicity and obvious in vitro and in vivo antimicrobial activity, which is expected to be an alternative treatment for drug-resistant S. aureus infection.