Abstract:[Objective] Control of the spread of tobacco black shank caused by Phytophthora nicotianae in tobacco production. [Methods] With Phytophthora nicotianae used as the assay fungus, bacterial strains antagonistic to P. nicotianae were isolated from tobacco rhizosphere soil obtained from Bijie District, Guizhou Province using dilution-plate methods and identified based on morphology, Biolog identification and 16S rRNA gene sequence analysis. The antimicrobial spectrum of these strains was further tested against Ralstonia solanacearum, Botrytis cinerea, Alternaria alternate and Colletotrichum destructivum in vitro. Optimization of growth conditions was also investigated through single factor variable analysis. [Results] Of 44 antagonistic strains obtained, strain 21b showed an inhibition rate of 78.33% to P. nicotianae and was identified as Bacillus subtilis. The size of inhibition zone of this strain against Ralstonia solanacearum, Botrytis cinerea, Alternaria alternate, and Colletotrichum destructivum were 19.5, 18.2, 14.6 and 13.4 mm, respectively. The optimal fermentation conditions for 21b was a temperature of 30 °C, pH 7.0?8.0, media/flask volume 12%, and 0.5% salt. [Conclusion] One bacterial strain with antagonistic activity against P. nicotianae isolated in this study could provide microbiological resources for the development of biocontrol of tobacco black shank in the future.