Abstract: Verticillium dahliae is a soil-borne, semi-living, vegetative, plant pathogenic fungus. Verticillium wilt, caused by V. dahlia, is one of the most important issues affecting cotton production worldwide. Root endophytes play an important role in plant resistance to pathogenic fungi. However, the impact of Verticillium wilt on the community composition and assembly of endophytic bacteria remains poorly understood. In this study, based on a long-term field experiment, changes in bacterial taxonomic composition, diversity, and community assembly processes in the roots of healthy and Verticillium wilt-diseased cotton plants were investigated using both cultivation-dependent and cultivation-independent technologies. High-throughput sequencing results showed that the diversity of the root endophytic bacteria was not significantly different between the two treatments. However, the taxonomic composition and community structure of the endophytic bacteria in diseased plant roots were significantly different from those in healthy plants. Compared with healthy plants, diseased plants contained significantly higher relative abundance of Proteobacteria, Bacteroidetota, and Firmicutes but lower abundance of Actinobacteriota, Verrucomicrobiota, and Myxococcota. Among the 4579 zOTUs detected in this study, 83% were shared between the two treatments; however, the relative abundance of these shared zOTUs was significantly different between the two treatments, which mainly contributed to the dissimilarity of bacterial communities between the two treatments. The biomarker species of the two treatments were identified using indicator species analysis (ISA). The healthy plants enriched zOTUs were assigned as Pseudomonas, Rhodocyclaceae, Moraxellaceae, and unidentified taxa in the roots, whereas the diseased plants enriched different zOTUs were assigned as Pseudomonas, Variovorax, Comamonadaceae, and Agrobacterium tumefaciens, among others. An antagonistic bacterial strain, ABLF-8 (designated Pseudomonas) against V. dahliae was detected in the roots of both healthy and diseased cotton plants. However, its relative abundance was significantly lower in diseased plants than in healthy plants, indicating that the antagonistic effect against V. dahliae in the root endophytic bacterial community of diseased cotton plants was lower than that in healthy plants. The results of the beta-NTI and beta-null model analyses showed that the deterministic process dominated the bacterial community assembly in cotton roots; however, its contribution was significantly lower in diseased plants than in healthy plants, indicating that the randomness of bacterial community assembly in diseased plant roots was higher than that in healthy plant roots, and the regulation of endophytic bacterial communities by plant roots was weakened by Verticillium wilt. In summary, the results of this study indicated that V. dahliae infection directly or indirectly reduced the effect of cotton on the assembly of bacterial communities in roots and caused a significant decrease in the relative abundance of antagonistic taxa against V. dahliae. This led to an imbalance of microbial flora in roots and reduced the antagonism of the endophytic microbial community against V. dahliae, which may be one of the important reasons for the further development of Verticillium wilt.