Abstract: In this study, actinomycetes were isolated from rhizosphere soil of tomato by spread plate method, with the pathogen Botrytis cinerea as the target fungus. An actinomycete isolate LA-5 with strong antifungal activity was obtained by confrontation culture and Oxford cup double screening method. According to cultural characteristics, physiological and biochemical properties and 16S rDNA analysis, the strain LA-5 was primarily identified as Streptomyces chungwhensis. The second screening results showed that the fermentation filtrate of LA-5 could substantially inhibit the spore germination and mycelium growth of B. cinerea. Both conidium germination and mycelia growth could be inhibited by 100 times fermentation broth of LA-5, with the inhibiting rate being higher than 50%. Furthermore, the inhibited colony showed white mycelia, with sparse and thin aerial hypha, and the obviously decreased branches. Results from in vitro biocontrol experiment showed that control efficiency of filtrate from LA-5 fermentation liquid on B. cinerea could be up to 83.4%, indicating that the strain was a potential antagonist against tomato gray mold.