Botrytis cinerea, the causal agent of gray mold, is a pathogen that attacks many crops. The local strain B. licheniformis B307 was previously selected as a possible control agent for this fungus through its chitinase production. The current study was conducted to characterize and purify chitinase produced from this strain, and determine the extract enzymatic antifungal inhibitory activity against gray mold fungus on tomato plant. Chitinase enzyme was produced from the B. licheniformis B307 strain by the submerged fermentation method, with an enzyme activity of 14 U/ml. The results of the partial purification showed that enzyme precipitation with 65% saturated ammonium sulfate was the best, since specific activity reached 1 U/mg compared to 0.24 U/mg for the raw extract. The results of the precipitated proteins electrophoresis showed that there were two fragments with a molecular weight of approximately 36 and 42 kDa associated with chinolytic activity. The results of genomic DNA amplification using CHI0 and CHI4 primers amplified a 1500bp band. The bio-assay results of antagonistic activity against B. cinerea on the three tomato varieties (Rome, Marmande, local) showed a reduction in disease incidence of 48.9-56.9% in the bacteria-treated plants and 40.66-50.87% in plants treated with enzymatic extracts, compared to the control. It can be concluded from this study that the bacterial strain B. licheniformis B307and its enzymatic extract can be an effective biocontrol treatment against gray mold, which makes this bacterial strain a potential alternative to chemical fungicides for the control of gray mold and possibly other pathogens.