Tobacco bacterial wilt caused great damage to tobacco production in China, especially in the southwest. It is a new approach to explore the use of the suppressive-soil to construct tobacco healthy rhizosphere soil for biological control of bacterial wilt, and it will provide a theoretical basis for novel biological control of tobacco. The disease-suppressive (hardly affected with disease) and -conducive soils (easily affected with disease) in Guizhou Province were used in seedling and potted simulation experiments. The treatments were designed as follows: Cn+Cp-conducive-soil in both seedling and pot experiments; Cn+Sp-conducive-soil in seedling and suppressive-soil in pot experiment; Sn+Cp-suppressive-soil in seedling and conducive-soil in pot experiment; Sn+Sp-suppressive-soil in both seedling and pot experiment. The high-throughput sequencing and Biolog technology were used to study the changes in the structure, function, and metabolic genes of soil microbial communities. The results obtained show that the control effect of Sn+Cp treatment was significantly higher than that of the Cn+Sp treatment. There was no significant difference of the functional diversity of soil microbial community between Sn+Cp and Cn+Sp treatments at 90 d. However, Gemmatimonadetes and Nitrospirae of the Sn+Cp treatment were significantly higher than those of Cn+Sp treatment, while Actinobacteria and Acidobacteria showed a reverse trend in Sn+Cp and Cn+Sp. The metabolic genes of general function prediction only, protein turnover and signal transduction mechanisms of the Sn+Cp treatment were up-regulated compared with those of Cn+Sp treatment, while transcription and carbohydrate transport and metabolism of Sn+Cp treated soil were down-regulated. Furthermore, there were more significant correlations between soil microorganisms and soil properties in the Sn+Cp treatment, compared with that in Cn+Sp treatment. In summary, the use of suppressive soil microorganisms in the seedling stage can construct healthy microbial communities in tobacco rhizosphere, thus reducing the occurrence of bacterial wilt. This result will provide new approaches for the biological control of tobacco bacterial wilt.