Abstract: Tomato grey mould, caused by Botrytis cinerea, is a severely damaging global disease. This study isolated a strain of antagonistic bacteria with strong inhibitory effects against the important plant pathogen Botrytis cinerea from healthy branches and leaves of Camellia sinensis in Yunnan's Gaoligong Mountain. The morphological and molecular biological identification results indicated that the antagonistic strain was Bacillus subtilis, which was designated as DB2203A. The fermentation conditions of strain DB2203A were optimized through single-factor and orthogonal experiments, and the antibacterial activity of the fermentation broth was determined along with a preliminary exploration of the antibacterial substances under these conditions. This study isolated and screened seven strains with strong antagonistic effects against pathogenic fungi from leaf and branch tissues. Among them, strain DB2203A exhibited the most significant inhibitory effect against B. cinerea, with its sterile fermentation broth achieving an inhibition rate of 74.77% against the pathogen. The optimal medium for the strain DB2203A was LB medium, with the best fermentation conditions being an inoculation volume of 6%, a filling volume of 50%, an initial pH of 7.0, and a fermentation time of 72 h. The research results showed that the antifungal activity of the 40 times diluted fermentation broth against B. cinerea is 59.25%. Moreover, the lipopeptide substances in the fermentation broth could inhibit the growth of the mycelium of B. cinerea. The strain B. subtilis DB2203A and its lipopeptide metabolites exhibit promising potential for the green control of tomato gray mold, providing a theoretical foundation and microbial resources for the development of microbial products.