Chaetomium subaffine LB‐1 is a biocontrol strain screened by our laboratory. Mycolytic effect and its action mechanism was researched in this study with Botrytis cinerea and Alternaria solani as test phytopathogenic fungi. Microscopic observation found that hyphae of the test fungi exhibited vacuolated and flocculent when cultured in LB‐1 culture filtrate for 48 h. Physio‐biochemical detection indicated that hyphae of the test fungi displayed red fluorescence with propidium iodide (PI) staining when cultured in LB‐1 culture filtrate 24 h later; conductivity and the leakage value of the extracellular fluid of the test fungi were significantly higher (p ≤ .05) than corresponding control fluid of test fungi cultured in potato dextrose broth (PDB), but the malondialdehyde (MDA) contents in hyphae of the test fungi have not significant difference (p ≤ .05) between treatment and control. Colorimetric determination showed that the activities of alkaline protease, neutral protease, acidic protease and β‐l,3‐glucanase in LB‐1 culture filtrate were significant. In RNA‐seq analysis, 3644 differentially expressed genes (DEGs) were found in 6 days shaking‐cultured LB‐1 as compared to that of 0 days shaking‐cultured, of which 1756 were up‐regulated and 1888 were down‐regulated. In significantly up‐regulated DEGs, 12 were annotated as cell wall hydrolase, 13 as enzyme of antibiotic substance production and 18 as membrane transporter. The above results indicated that C. subaffine LB‐1 might exert mycolytic effect through altering the permeability of cell membrane and the integrity of cell wall of phytopathogenic fungi, and this altering effect was closely related with cell wall hydrolase, antifungal substance and membrane transporter.