Tobacco bacterial wilt (TBW) is caused by bacterium Ralstonia solanacearum. To effectively develop microoganisms for biocontrol of TBW, 150 strains of tobacco endophytic bacteria from the stems of the health plant at TBW areas in Chenzhou, Yongzhou of Hunan were obtained by the dilution method. Antagonistic test showed 21 of the 150 strains, accounting for 14%, had antagonistic effects against R. Solanacarum. Among the 21 strains, strain F1 showed the best antagonistic effect with the diameter of inhibition zone against tobacco R. solanacarum strain 2316 at 24 hours of 6.5 mm, and showed a wide antimicrobial spectrum including five kinds of pathogenic fungi such as Sclerotium rolfsii and five kinds of pathogenic bacteria such as Xanthomonas campestris pv. citri. Based on analysis of the morphological, physiological, and biochemical characteristics and the 16S rDNA phylogeny, strain F1 was identified as Bacillus mojavensis.