Soil microbial community composition of crop rhizosphere is very important in soil ecology and crop health. In this paper, the soil with different levels of cotton Verticillium wilt is the research object. Moreover, physical and chemical properties of soil, and rhizosphere microbial community diversity were analyzed with the technique of microbial pure culture, physical and chemical analysis and high-throughput sequencing. The results indicate that sterile fermentation liquor of Verticillium dahliae has obvious inhibitory effect on bacterial growth in pure culture conditions. After inoculation with V. dahliae, the quantity of bacteria, fungi, and actinomycetes in the cotton soil show little change compared to that in the control. High-throughput sequencing analysis indicates that bacterial genetic relationship in soil shows no significant change, and sampling time has a bigger impact on the bacterial population structure. Soil fertility plays a key role in determining the quantity of bacteria, fungi, and actinomycetes in continuous cotton cropping soil, in which Verticillium wilt occurrence is severe and significantly higher than that in the control field. Rice-cotton rotation cropping has a strong effect on reducing salt, plant diseases, and improving soil fertility. Microsclerotia content in soil has significant positive correlation with the incidence of cotton Verticillium wilt. Soil fertility, culture measures, and crop varieties mainly influence soil microorganism quantity; however, Verticillium wilt occurrence does not cause significant change in microbial quantity. Reducing the quantity of microsclerotia in soil is the fundamental method of controlling Verticillium wilt.