Prokaryotic communities play a pivotal role in maintaining ecosystem balance through their involvement in essential processes including carbon fixation, nutrient cycling, and the decomposition of organic matter. Despite the importance of prokaryotic communities in wastewater treatment plants and rivers, their diversity and distribution in these environments in Egypt are not well understood. To bridge this gap, the study utilized next-generation 16S rRNA amplicon sequencing based on PacBio technology to investigate the composition and diversity of these microbial communities in wastewater treatment plant (inlet and outlet) and the Nile River. The principal coordinate analysis showed that the microbial community structure varied significantly between the three habitats, indicating the wastewater treatment process effectively removes pollutants and facilitates the growth of diverse microbial communities. Proteobacteria increased in outlet and surface water (>50%) versus inlet (45%), while Actinobacteria increased in surface water (>20%). Firmicutes and Campilobacteria decreased significantly (P≤0.05) in outlet versus inlet, and Chloroflexi were only found in outlet (<2%). Environmental factors such as EC and NH₄-N were the most significant variables in explaining the variation in microbial communities. It’s worth noting that the final effluent from the Zenin WWTP aligns with the standards set by the Egyptian ministerial decree 48/1982 for discharging in surface water bodies. Understanding the composition of microbial communities is critical for maintaining ecosystem function, including nutrient cycling and decomposition of organic matter.