The tailwater from wastewater treatment plants serves as an important water resource in arid regions, alleviating the conflict between supply and demand. However, the effects of different tailwater discharge proportions on microbial community dynamics remain unclear. In this study, we investigated the effects of different tailwater discharge proportions on the water quality and microbial community characteristics of sediments in receiving water bodies under controlled conditions (WF-1, WF-2, WF-3, WF-4, and WF-5, containing 0% tailwater + 100% natural water, 25% tailwater + 75% natural water, 50% tailwater + 50% natural water, 75% tailwater + 25% natural water, and 100% tailwater + 0% natural water, respectively). Microbial co-occurrence networks and structural equation model were used to unveil the relationship between microbial communities and their shaping factors. Results showed that distinct microbial community compositions were found in the sediments with low- (< 50%) and high- (> 50%) proportions of tailwater. Specifically, WCHB1-41 and g_4-29–1, which are involved in organic degradation-related functions, were the key genera in the high-proportion cluster. A total of 21 taxa were more abundant in the low-proportion (< 50%) cluster than that in high-proportion (> 50%). Moreover, higher modularity was observed in the low-proportion. Total phosphorus directly affected while ammonia nitrogen indirectly affected the microbial community structure. Our findings support the distinct heterogeneity of microbial communities driven by tailwater discharge in receiving water bodies, and further confirmed that high-proportion tailwater depletes sensitive microbial communities, which may be avoided through scientific management.