Inter-basin water transfer is an effective manner to achieve the optimal allocation of water resources, while accompanied by some ecological effects. The responses of microorganisms to water diversion and the ecological processes in regulating the community assembly are still unclear. Taking the eastern route of South-to-North Water Diversion Project as the study area, we investigated the microbial community patterns and the underlying assemblage processes in habitats with different hydrological connectivity, including isolated lakes, connected lakes and man-made canal. The results showed that microbial communities in the canal had higher diversity, lower dissimilarity, weaker compositional variation, and stronger co-occurrence patterns compared with that in the connected and isolated lakes. These findings suggested that the increase of connectivity among natural aquatic habitats due to water diversion can homogenize microbial communities and reduce microbial heterogeneity. The neutral and null models demonstrated the importance of stochastic processes in shaping microbial community assembly. Dispersal limitation and variable selection were the predominant mechanisms structuring microbial communities in the isolated lakes. Due to the homogenized environmental condition and the enhanced hydrologic connectivity in the canal and the connected lakes, microbial communities had higher dispersal capability and ecological drift occurred more frequently in these lotic habitats. The variations in microbial community structure were mainly driven by biotic ecological succession than abiotic factors, with positive and negative cohesion explained 63% and 25% of variability, respectively. Six taxa were considered as the potential introduced microorganisms, which may favor the nutrient biogeochemical cycling and the organic matter degradation, but may also bring ecological risks. Overall, this study provides a deeper understanding of the ecological consequences of inter-basin water diversion, and helps the regulation and management of these projects.