Abstract: AIM: To discover actinomycete strains-producing analogs and structure-types with anti-methicillin-resistant Staphylococcus aureu (anti-MRSA) activity from a soil used as an anti-infection folk medicine. METHODS: Plate method was used for the isolation of actinomycete strains with an improved Gauss medium, and chemical screening of these strains was achieved with HPLC-UV technique. The anti-MRSA activities and minimum inhibitory concentrations (MICs) of the extracts of the cultures of target strains were performed by agar diffusion and broth microdilution respectively, and their cytotoxicity was evaluated by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) method using human colon tumor cell HCT-116. RESULTS: Sixty-one actinomycete strains were isolated from the soil sample collected in Zhuzhou County, Hunan, China. Based on the analogs and structure- types in the HPLC profiles of the extracts of their cultures, three strains ZZ027, ZZ021 and ZZ044 were targeted by chemical screening. The extract (500 g) of the culture of ZZ027 and ZZ021 showed anti-MRSA activity with inhibition zones of 2935 and 1724 mm respectively, and their MICs were 0.6251.25 and 2.50 mgmL1 respectively. The extract obtained from ZZ021 showed remarkable cytotoxicity against HCT-116 in vitro with IC50 value of 6.4 gmL1, while those of ZZ027 and ZZ044 showed no cytotoxicity (IC50 values were more than 20 gmL1). CONCLUSION: From a soil sample used as an anti-infectious folk medicine, three strains ZZ027, ZZ021 and ZZ044 were obtained by selective isolation and chemical screening. ZZ027 and ZZ021 showed anti-MRSA activities, and ZZ021 showed remarkable cytotoxicity.