Abstract: 【Objective】 This study aimed to investigate the main antibacterial substances derived from Bacillus safensis strain GX-H6， and to screen the optimal fermentation culture conditions to maximize the yield of antibacterial substances， so as to provide a foundation and materials for subsequent theoretical studies and applications in the prevention and control of rice bacterial leaf streak using the GX-H6 strain. 【Method】 Utilizing the previously isolated Bacillus safensis GX-H6 strain as the test strain， and Xanthomonas oryzae pv. oryzicola GX01 as the indicator， antibacterial compounds were extracted from GX-H6 using methanol， ethanol， water， ethyl acetate， and chloroform. The extracts were subsequently analyzed via liquid chromatography-mass spectrometry （LC-MS） to identify their constituents. Carbon and nitrogen sources， the type and concentration of inorganic salts， fermentation time and temperature， and initial pH of the initial fermentation media for the GX-H6 strain were optimized through single-factor experiments. Then the optimal culture conditions were validated using an orthogonal experimental design. 【Result】 The antibacterial substances produced by the strain GX-H6 were extracted with methanol， and the crude extract of the antibacterial substance produced an inhibition zone of 16-18 mm in diameter against strain GX01 in the Oxford cup assay. According to the LC-MS analysis， the two antibacterial substances with their mass-to-charge ratio of 1036.69 and 1050.70 m/z were probably surfactins. Through the single-factor and orthogonal test， a high-yield culture medium formulation was optimized， containing 5.0 g/L trehalose， 12.0 g/L yeast extract， 1.0 g/L NaCl， and an initial pH of 8.0； the optimized culture condition was 37 ℃ for 48 h. The antibacterial zone diameter increased 2.4-fold compared to that before optimization. 【Conclusion】 The primary antibacterial substance produced by Bacillus safensis GX-H6 is probably a surfactin. The optimized high-yield medium formulation and fermentation conditions established in this study enhance efficient production of surfactin. As an environmentally safe biocontrol agent， the GX-H6 can be produced cost-effectively and locally with existing technology， demonstrating its significant potential to be a biocontrol bacterium against rice bacterial leaf streak.