[Objective] To study the diversity and antimicrobial activity of culturable actinomycetes isolated from Apis cerana cerana Fabricius, which will help to explore new microbial resources. [Methods] Seven media were used to isolate actinomycetes from Apis cerana cerana Fabricius samples. 16S rRNA PCR-RFLP analysis and 16S rRNA gene sequences analysis were utilized to study their genetic diversity. Pathogenic bacteria and fungus were used in antimicrobial test for exploring the antimicrobial activity of strains. [Results] Based on the morphology of the colonies and cells of the new isolates, 84 representative strains were selected from 180 isolated strains. They belonged to 3 orders, 4 families and 4 genera, and in which there were 6 strains identified as potential new novel species. The best surface disinfection method was by using 0.2% ClO2 for 60 s. The results of the antimicrobial test showed that 31.0%, 48.8%, 27.4% and 16.7% of the representative strains presented antimicrobial activity to Escherichia coli, Bacillus subtilis, Curvularia lunata and Fusarium oxysporum, and 71.4% of the representative strains presented antimicrobial activity to at least one pathogen. [Conclusion] The choice of the isolation method has a great impact on isolation result. There are great diverse actinomycetes inside Apis cerana cerana Fabricius and they have great antimicrobial activity, which show a great potential for researching new bioactive compounds.