Abstract: Bacillus subtilis Bs916 is an effective biocontrol agent against rice sheath blight caused by Rhizoctonia solani. The lipopeptide bacillomycin L plays a crucial role in biocontrol activities of strain Bs-916. In this study, bcillomycin L was isolated and purified from the culture of B. subtilis Bs-916 and in vitro assay indicated that bacillomycin L was toxic to all the 12 tested pathogenic fungi and IC₅₀ values varied from 0.2 μg·mL^-1 to 40 μg·mL^-1. Magnaporthe oryzae and Botrytis cinerea showed the highest sensitivity to bacillomycin L and IC₅₀ values on hyphal growth were 0.35 μg·mL^-1 and 0.28 μg·mL^-1, respectively. For most of the tested fungi, IC₅₀ values were 1-3μg·mL^-1. Fusarium fujikuroi and F. moniliforme showed the lowest sensitivity to bacillomycin L and IC₅₀ values were 28.99 μg·mL^-1 and 45.92 μg·mL^-1, respectively. Microscopic observation demonstrated that the hyphae of the pathogenic fungi treated with bacillomycin L showed abnormal growth, and conidia produced enlarged and constricted germ tube. Cellular leakage was also observed when bacillomycin L was used in high concentration. With 500 μg·mL^-1 bacillomycin L,the control efficiency against rice sheath blight and rice seedling blast reached 70.8% and 56.3%, respectively. Results above indicated that bacillomycin L has a broad antifungal activity and potential application in biological control.