The connection between microbial community structure and spatial variation and pollution in river waters has been widely investigated. However, water and sediments together have rarely been explored. In this study, Illumina high-throughput sequencing was performed to analyze microbes in 24 water and sediment samples from natural to anthropogenic sources and from headstream to downstream areas. These data were used to assess variability in microbial community structure and diversity along in the Fenghe River, China. The relationship between bacterial diversity and environmental parameters was statistically analyzed. An average of 1682 operational taxonomic units was obtained. Microbial diversity increased from the headstream to downstream and tended to be greater in sediment compared with water. The water samples near the headstream endured relatively low Shannon and Chao1 indices. These diversity indices and the number of observed species in the water and sediment samples increase downstream. The parameters also differ in the two river tributaries. Community structures shift based on the extent of nitrogen pollution variation in the sediment and water samples. The four most dominant genera in the water community were Escherichia, Acinetobacter, Comamonadaceae, and Pseudomonas. In the sediments, the most dominant genera were Stramenopiles, Flavobacterium, Pseudomonas, and Comamonadaceae. The number of ammonia-oxidizing archaea in the headstream water slightly differed from that in the sediment but varied considerably in the downstream sediments. Statistical analysis showed that community variation is correlated with changes in ammonia nitrogen, total nitrogen, and nitrate nitrogen. This study identified different microbial community structures in river water and sediments. Overall this study emphasized the need to elucidate spatial variations in bacterial diversity in water and sediments associated with physicochemical gradients and to show the effects of such variation on waterborne microbial community structures.