Abstract:[Objective] Screening the antagonistic endophytic bacteria against Thielaviopsis basicola from inner of tobacco roots and stems collected from Chongqing. [Methods] The antagonistic effect of 306 endophytic bacteria against Thielaviopsis basicola by pairing culture on PDA plates, the strain was identified by morphological, physiological and biochemical characteristics, and homology analysis of the partial sequence of 16S rDNA. [Results] Six endophytic bacteria were showed high antagonistic effect against Thielaviopsis basicola, the pathogen of tobacco black root rot. The inhibition zone diameters were ranged from 6.5 mm to 11.0 mm by pairing culture on PDA plates, and the control efficacy ranged from 11.9% to 77.1% in a greenhouse experiment. It was found that strain T295 could reduce the incidence of tobacco black root rot, the control efficacy reaching to 77.1%, and the culture filtrate of strain T295 could inhibit hyphal growth and reduce spore germination of T. basicola. [Conclusion] Strain T295 had a good antagonistic effect on Thielaviopsis basicola, was identified as Bacillus subtilis.