Bacteria and microeukaryotes inhabiting urban freshwater are susceptible to environmental disturbance, particularly effluents from wastewater treatment plants (WWTPs). However, how riverine microbial community succession responds to WWTP effluent and its assembly processes remains poorly addressed. Herein, surface water samples were collected from an urban river to explore the community dynamics and assembly processes under the impact of the WWTP effluent. Physicochemical property variation (e.g., total phosphorus and nitrate concentration) caused by wastewater was more strongly correlated with the microbial community than with geographic distance. Importantly, deterministic processes increase from 34.8 to 93.3% for bacterial community assembly from upstream to downstream. Betaproteobacteria, Bacillariophyta, and Chlorophyceae exhibited essential roles in mediating the community composition against WWTP effluent discharge. In addition, bacteria tend to have a stronger interaction in wet or normal periods, while microeukaryotes may have more stable networks in dry periods. Seasonal variation caused a significant discrepancy (ANOVA P = 0.001) in community composition for both bacteria and microeukaryotes. Our work provides systematic information on how treated wastewater contributes to microbial community succession and identifies the potential keystone taxa during the microbial response in wastewater, which may lead to improved management of wastewater discharge and river health.