Although the influence of microbial community assembly processes on aquatic ecosystem function and biodiversity is well known, the processes that govern planktonic communities in human-impacted rivers remain largely unstudied. Here, we used multivariate statistics and a null model approach to test the hypothesis that environmental conditions and obstructed dispersal opportunities, dictate a deterministic community assembly for phytoplankton and bacterioplankton across contrasting hydrographic conditions in a subtropical mid-sized river (Jiulong River, southeast China). Variation partitioning analysis showed that the explanatory power of local environmental variables was larger than that of the spatial variables for both plankton communities during the dry season. During the wet season, phytoplankton community variation was mainly explained by local environmental variables, whereas the variance in bacterioplankton was explained by both environmental and spatial predictors. The null model based on Raup-Crick coefficients for both planktonic groups suggested little evidences of the stochastic processes involving dispersal and random distribution. Our results showed that hydrological change and landscape structure act together to cause divergence in communities along the river channel, thereby dictating a deterministic assembly and that selection exceeds dispersal limitation during the dry season. Therefore, to protect the ecological integrity of human-impacted rivers, watershed managers should not only consider local environmental conditions but also dispersal routes to account for the effect of regional species pool on local communities.