Objective To explore the diversity of endophytic and habitat soil fungi of Cordyceps militaris in different regions and conduct antibacterial activity analysis, and to screen out strains with good antibacterial activity. Methods In this study, the symbiotic microorganisms of C. militaris were isolated by pure culture methods, and the antibacterial activity of endophytic and habitat soil fungi of C. militaris was tested by the plate confrontation method. Results A total of 86 endophytic fungi belonging to 4 phyla, 22 genera and 51 species and 71 soil fungi belonging to 4 phyla, 22 genera and 46 species were obtained from C. militaris. The dominant genera of endophytic and habitat soil fungi in the four regions of C. militaris were Aspergillus, Fusarium and Trichoderma. Conclusion This study confirmed the rich diversity of symbiotic microorganisms of different populations of C. militaris by pure culture methods. The active strains screened provide a basis for the development and utilization of endophytic and soil fungal resources of C. militaris, and have certain significance for the protection of natural Cordyceps species and Cordyceps resources.