Botrytis cinerea causes considerable economic losses to the tomato processing industry. Hence, the study evaluated the antifungal activity of eight biocontrol strains isolated from tomato rhizosphere soil against B. cinerea. These strains demonstrated potent antifungal activity with an inhibition rate ranging from 19.30% to 69.88%. Among them, a strain named D50 showed the highest effect with an inhibition rate of 69.88%. D50 strain was identified as Bacillus mojavensis by morphological, physiological, and biochemical analyses. The Bacillus mojavensis D50 fermentation supernatant (BMFS) was used for subsequent experiments to unveil antimicrobial activity of its secondary metabolites. BMFS (5%, v/v) reduced the growth of B. cinerea by inhibiting mycelial growth, conidial production, and mycelial dry weight by 51.7%, 45.9%, and 53.7%, respectively. Detailed analysis revealed that the antifungal effect of BMFS decreased when it was heated above 80 °C and adjusted to pH 2, 8, and 10. The antifungal effect also decreased when exposed to UV for more than 40 min. Further, the in vivo effect of BMFS in preventing gray mold was evaluated using cherry tomatoes. After B. cinerea inoculation for 5 days, the disease incidence and disease severity of cherry tomato treated with BMFS decreased by 33.5% and 31.5%, respectively. BMFS treatment enhanced the activities of peroxidase (POD), catalase (CAT), polyphenol oxidase (PPO), superoxide dismutase (SOD), and phenyl alanine lyase (PAL) and increased the content of malondialdehyde (MDA) in cherry tomatoes during infection. Moreover, BMFS prevented tomato gray mold by enhancing the transcript levels of systemic acquired resistance-related marker genes (SlLoxD, SlMyc2, SlPR1-a, SlPAL5, SlEIN2, and SlEIN3). SlLoxD and SlMyc2 had high expression levels among the defense-related genes, indicating activation of the jasmonic acid signal pathway. Thus, the study demonstrates the great potential of BMFS in managing the postharvest gray mold of tomato caused by B. cinerea.