Municipal wastewater treatment plants (WWTPs) are crucial for protecting the environment and public health, yet the discharge of treated wastewater can influence the biodiversity of aquatic microbial communities. Enterobacterales are reliable indicators of sanitary risk. Contamination with Enterobacterales often reflects wastewater treatment inefficiency, and pathogenic strains such as E. coli, Klebsiella pneumoniae, and Enterobacter pose significant public health threats. This study assessed bacterial diversity in the wastewater treatment process and evaluated how treated wastewater affects the microbiome of the Pilica River. Its added value lies in the use of an integrated catchment-scale approach, involving an analysis of the Pilica River from its source to its mouth (including eight sampling sites), all seasons, and inflows from 17 WWTPs. The abundance of Enterobacterales was strongly correlated with environmental factors, but not with pH. WWTP size influenced the relative abundance of ASVs of Yersinia, Escherichia-Shigella, and total Enterobacterales, while influent composition had no significant effect on microbial communities. Seasonal variations had the greatest impact on river microbiota, particularly Yersinia, Rahnella, and Providencia. Escherichia-Shigella dominated across wastewater and river samples, confirming its role as an indicator of water quality. The study demonstrated that treated wastewater can modify river microbiomes, thereby increasing sanitary and epidemiological risks.