Abstract: Dictyophora rubrovalvata, as an edible mushroom with high economic value, faces severe constraints in its large-scale production due to frequent disease outbreaks. Identifying the pathogenic species and screening highly effective fungicide combinations are of great significance for effectively controlling this disease. In this study, tissue isolation and in vitro wound inoculation were performed on diseased parts of D. rubrovalvata collected from planting bases in Zhijin County, Guizhou Province. The isolated pathogens were identified using morphological characteristics combined with ITS sequence-based phylogenetic analysis. The mycelial growth rate method was employed to systematically evaluate the biological activity of 22 selected pesticide formulations, initially screened as single agents and then as combined mixtures. The most synergistic combination and optimal ratio were identified, and their efficacy was further investigated through field trials. The results showed that the isolated pathogen was Munro hemisphaericum. The combination of 40% difenoconazole suspension concentrate (SC) and 0.3% tetramycin (AS) at a volume ratio of 1∶6 exhibited the most prominent synergistic effect, with a synergy ratio (SR) of 1.75. Field trials demonstrated that treatment with 100 mg/L of the combined fungicide achieved a control efficacy of 80.39%, indicating satisfactory disease management. This study provides a reference for the rational use of fungicides to control diseases in D. rubrovalvata caused by M.hemisphaericum.