Bacterioplankton play critical roles in biogeochemical cycling. Although spatial and temporal variations in bacterioplankton community compositions (BCCs) within individual habitat have been reported, knowledge gaps remain for studies conducted within different habitats. In this work, we examined the seasonal and spatial variability of BCCs in Nanfei River and Lake Chaohu which had significant environmental heterogeneity using a high-throughput sequencing technique of 16S rRNA gene amplicons. The results showed that spatial variation has a more obvious impact on the BCCs than seasonal changes. The microbial diversity gradually decreased and BCCs changed obviously along water flow direction from Nanfei River to the western and estern parts of Lake Chaohu over all seasons. LEfSe analysis showed that Nanfei River had higer abundance of species belonging to the orders Rhodocyclales, Methylococcales, Campylobacterales and Flavobacteriales, samples from eastern part of Lake Chaohu were abundant in taxonomies including the order Rickettsiales, while the western part had high abundance of taxonomies belonging to the order Chroococcales. The redundancy analysis (RDA) indicated that BCCs in Nanfei River were associated with high nutrient (TP, PO4-P, TN, NH3-N, NO2-N and NO3-N) concentrations and electrical conductivity. Variance partitioning RDA analysis indicated that the combined effects of all variables may be most important affecting BCCs. This study may provide a framework for modeling the change in bacterioplankton communities through different habitats from a river to lake.