Contaminated samples or fruiting bodies of Hypsizygus marmoreus were collected from 3 provincial producing bases subordinated to a large-scale mushroom production enterprise in the whole year of 2018 for investigation of pollution in mushroom production. Molecular sequencing and morphological observation were made for identification of 11 isolates of microorganisms, and the influence of the microorganisms on the growth of the hyphae of Hypsizygus marmoreus were analyzed by plate confrontation experiment. The 11 isolates of microorganisms could be assigned to 3 families and 7 genera, of which 10 isolates were bacteria and 1 was a species of fungi. Enterobacteriaceae was the dominant family and Bacillus was the dominant genera. Two isolates of bacteria respectively belonging to Bacillus and Enterobacter were possibly new species. Results of plate confrontation experiment showed that 7 bacteria and 1 fungus significantly inhibit mycelial growth of H. marmoreus. It is proposed that substrate preparation, sterilization and fruiting management should be regarded as 3 critical control points for disease prevention in H. marmoreus cultivation. During low temperature period in autumn and winter, the substrate sterilization and temperature-raising time should be properly prolonged. In summer when the cultivation room needs to cool down using equipment, strict management of cultivation environment during fruiting period can helps reduction of the incidence of disease.