A total of 900 rhizosphere soil samples were collected from the main tobacco producing areas in Hunan Province and 195 bacterial groups with antagonistic effect on tobacco black shank were screened out after enrichment and cultivation. The high-throughput sequencing results of Miseq showed that Acinetobacter and Stenotrophlomonas are the common dominant genera of antagonistic bacteria. Five bacteria groups with better bacteriostatic effect were selected and applied to the soil of tobacco field, the control effect of group B44R on tobacco black shank was 51.56%. Antagonistic strains in the 5 bacteria groups were screened and isolated, the obtained antagonistic strains were found to belong to 4 genera: Bacillus, Acinetobacter, Stenotrophomonas and Klebsiella. The results of soil high-throughput sequencing showed that after applied to soils, the antagonistic bacteria could colonize in the soil and the populations of Acinetobacter and Bacillus increased significantly.