Root-knot nematode (RKN) disease is a soil-borne disease. However, most studies on RKN have focused on the screening of agents and the cultivation of resistant varieties, and reports on the interaction of RKNs with soil microorganisms are few. In this study, we performed Illumina high-throughput sequencing to analyze diseased and healthy soil and the microbial-community changes in rhizosphere soil after microbial treatment (Pseudomonas flurescens, Bacillus subtilis, Paecolomyces lilacinus). Results showed significant differences in the bacterial community richness and diversity between diseased and healthy soil and the presence of different microbial species. After treatment, the richness and diversity of microbial communities in soil, as well as the number and incidence of second-stage juvenile of RKNs, decreased. Through linear discriminant analysis effect size, Pearson correlation, and Venn diagram analysis, we screened five genera that were closely related to disease occurrence, among which Pseudomonas was most related to disease inhibition. Our results suggested that the occurrence of tobacco RKN was related to changes in soil microbial communities, and that the interactions among Pseudomonas, Bryobacter, Variibacter, Coniochaeta, and Metarhizium affected the health of rhizosphere soil.