The use of antagonistic microorganisms in the suppression of phytopathogenic fungus represents the ultimate approach to biological control, and because of their significant beneficial effects, antagonistic microorganisms are increasingly being researched. In order to reduce the use of agrochemicals, the discovery of antagonistic microorganisms and their pathogen supressive mechanisms are necessary in modern plant production. The necrotroph fungus Botrytis cinerea known as the causative agent of gray mold, leads to significant global losses of yield and production quality in the field and in post-harvest storage. Due to the significant arsenal of toxins used in pathogenesis and great genetic variability, this fungus is considered a pathogen of high risk for the development of fungicide resistance, which is why the use of antagonistic microorganisms in pathogen suppression is a promising strategy. Among the antagonistic microorganisms of biological control, the entomopathogenic fungus Beauveria bassiana is mentioned, which is considered to have antagonistic properties in controlling phytopathogens due to the wide range of secondary metabolites. In order to maximize the effectiveness of antagonistic fungi in field conditions, in recent laboratory studies, special attention is paid to researching the antagonistic mechanisms of fungi under optimal conditions for their growth and development which vary depending on the isolates of the same species. Based on the above, the antifugal effect of the fungus B. bassiana on the phytopathogen B. cinerea was tested in vitro using the dual cultures method and testing the production of volatiles on a standard PDA substrate with two different pH values (pH= 5.6 and 7.2). The results of dual culture tests and volatile production testing confirmed that the antagonistic effect of the fungus B. bassiana is significantly higher on a neutral pH substrate (pH=7.2), where the inhibition of the B. cinerea was 35.9 %, i.e. 37.5 % which was achieved by different antagonistic mechanisms. On the substrate with a moderately acidic pH (pH= 5.6), the percentage of inhibition achieved was about 8 (4.4 %), i.e. 3 (12.5 %) times lower compared to the inhibition achieved on the substrate with a neutral pH value. Microscopic analysis confirmed inhibition mechanisms (mycoparasitism and antibiosis), more significant on a neutral pH substrate by the antagonistic species B. bassiana, and deformations and changes in the microstructures of the pathogen B. cinerea. According to the obtained results, the isolate of entomopathogenic fungus B. bassiana has a significant antagonistic potential in suppressing the causative agent of gray mold in laboratory conditions, which is the starting point for further research in vivo.