The characterization of variations in riverine microbiota that stem from contaminant sources and transport modes is important for understanding biogeochemical processes. However, the association between complex anthropogenic nitrogen pollution and bacteria has not been extensively investigated owing to the difficulties faced while determining the distribution of nitrogen contaminants in watersheds. Here, we employed the Soil and Water Assessment Tool alongside microbiological analysis to explore microbial characteristics and their responses to complex nitrogen pollution patterns. Significant variations in microbial communities were observed in sub-basins with distinct land–water pollution transport modes. Point source-dominated areas (PSDAs) exhibited reduced microbial diversity, high number of denitrification groups, and increased nitrogen cycling compared with others. The negative relative deviations (−3.38) between the measured and simulated nitrate concentrations in PSDAs indicated that nitrate removal was more effective in PSDAs. Pollution sources were also closely associated with microbiota. Effluents from concentrated animal feeding operations were the primary factors relating to the microbiota compositions in PSDAs and balanced areas. In nonpoint source-dominated areas, contaminants from septic tanks become the most relevant sources to microbial community structures. Overall, this study expands our knowledge regarding microbial biogeochemistry in catchments and beyond by linking specific nitrogen pollution scenarios to microorganisms.