Although reclaimed water (RW) has become a promising alternative source for alleviating water shortage in arid and semiarid regions, the ecological risks it poses to the receiving water bodies remain largely unknown. Dissolved organic matter (DOM) is crucial for affecting the quality of RW and strongly influences bacterial communities (BCs) in aquatic ecosystems. In this study, we aimed to unravel the role of DOM signatures on the spatiotemporal composition of microbial communities (MCs) in a seasonally ice-sealed urban river that had been chronically replenished by RW. We found that discharging RW resulted in elevated DOM levels in the receiving river. Notably, an increase of 10% in protein-like substances was revealed. The differences between compositional characteristics of DOM and the abundance of riverine BCs between freezing and non-freezing periods were revealed. In the freezing season, humic-like components, aromaticity, and hydrophobicity of DOM were more significant, and bacterial taxa such as Bacteriodetes and Flavobacterium were increased, while Proteobacteria was decreased. Similarly, co-occurrence network analysis revealed an enhanced interplay between DOM and BCs at the same time. However, Klebsiella pneumoniae markedly decreased during the ice-sealed period. These results suggest that variations in DOM characteristics have remarkable impacts on the dynamics of aquatic BCs, which points to the need for a DOM−oriented RW quality monitoring strategy.