Bacteria and micro-eukaryotes play important roles in river ecological systems. The processes that govern bacterial and micro-eukaryotic communities in urban rivers are still uncertain. The spatiotemporal characteristics and assembly processes of bacterial and micro-eukaryotic communities in the Xiangjianghe River were explored using 16 S and 18 S rRNA gene amplicon sequencing in the present study. The results indicate that the bacterial and micro-eukaryotic community composition exhibited distinct temporal and spatial variation. The topological characteristics of co-occurrence networks demonstrate that the bacterial and micro-eukaryotic community coexistence patterns vary significantly between the four seasons. Water temperature (WT) and oxidation-reduction potential (ORP) were detected as the most critical factors affecting bacterial and micro-eukaryotic community structure. The stochastic process (dispersal limitation) was the dominant assembly process for bacteria and micro-eukaryotes in all seasons. Deterministic and stochastic processes influenced the bacteria and micro-eukaryotes differently. Compared to bacteria, the values of niche breadth were relatively lower, and the proportion of deterministic processes was relatively higher in micro-eukaryotes. These results expand our understanding of spatiotemporal patterns, assembly mechanisms, and influencing factors of bacterial and micro-eukaryotic communities in urban rivers.