Abstract: The activation experiments of twelve natural inactive actinomycetes by inducing streptomycin-resistance mutation were carried out.Ninety-nine mutants were obtained at streptomycin concentration of 25 μg/mL (MIC).Seven mutants showed bioactivity against tested bacterial were screened out,and one of them—the strain 67S-14 exhibited strong and constant inhibition effect.The results of antifungal activity in vitro showed that the inhibitory rate of fermentation broth of the strain 67S-14 was more than 50% higher than that of the wild strain No.67 against Exserohilum turcicu,Botrytis cinerea and Fusarium oxysporum f.sp.vasinfectum.The mutant 67S-14 showed obvious differences in the morphological characteristics compared with the wild-type strain No.67.The results of paper layer chromatography demonstrated that the bioactive substance might be a macrolide antibiotic.HPLC analysis showed that the antibiotic might be a metabolite of mutant 67S-14.