Soil microorganism has a profound influence on planting growth and disease suppression. However, the difference in microbial community structure between suppressive and root rot-conducive soil and the mechanism of controlling soil-borne diseases by microorganisms in suppressive soil were not clear. To provide a theoretical foundation for prevention and control of root rot, this paper investigated the change of community structure in rhizosphere soil between suppressive and root rot-conducive tobacco fields. Soil samples were collected during before transplanting, vigorous growing period, and mature period of the tobacco, and bacteria and fungi were analyzed using 16S rRNA and 18S rRNA gene sequencing, respectively. Results showed that bacteria were more sensitive to the change between suppressive and root rot-conducive soil, and fungi were more sensitive to the change of different tobacco growth periods. Compared with conducive soil, tobacco suppressive soil can resist the invasion of pathogens, especially fungi, by regulating soil microbial community structure, and the potential pathogen Boeremia was always lower. Fusarium, the root rot pathogen, decreased rapidly in the mature period in suppressive soil. Moreover, norank_o_Gaiellales and unclassified_f_Trichocomaceae had a critical role in suppressive soil in the process of inhibiting root rot, which was obvious in the mature stage. Overall, the results indicated that the composition and structure of the microbial community significantly altered between suppressive and conducive soil along with the growth of tobacco, and suppressive soil could inhibit the occurrence of soil-borne diseases by boosting beneficial bacteria and inhibiting the potential pathogens.