Human activities have significantly altered the biogeochemical cycles of carbon, nitrogen, and sulfur in aquatic ecosystems, leading to ecological problems.This study utilized 16S rRNA gene high-throughput sequencing and excitation-emission matrix parallel factor analysis (EEM-PARAFAC) to evaluate the bacterial community composition and dissolved organic matter structure in the upstream (less impacted) and downstream (severely impacted) sections of the river, with a focus on the interactions between bacterial diversity and dissolved organic matter (DOM) characteristics.Results indicated significant spatial diversity in bacterial communities, with a higher α-diversity upstream compared to the more polluted downstream sections. Environmental parameters, particularly total phosphorus (TP) and dissolved oxygen (DO), were found to significantly influence the distribution and composition of bacterial phyla through redundancy analysis. The pattern of bacterial community assembly has shifted from predominantly deterministic to predominantly stochastic as a result of human activities. The analysis of DOM through EEM-PARAFAC identified three main fluorescent components, reflecting varied sources and interactions with bacterial communities. Upstream, microbial activities predominantly contributed to autochthonous DOM, while downstream, increased inputs of allochthonous DOM from human activities were evident. Furthermore, the study revealed that through the introduction of various organic pollutants and nutrient loads that shift microbial metabolic functions towards increased degradation and transformation of complex organic compounds downstream. Structural equation modeling (SEM) revealed that upstream human activities primarily affected bacterial communities indirectly by altering DOM properties. In contrast, downstream activities had both direct and indirect effects due to higher pollutant loads and more complex environmental conditions. These interactions underline the profound effect of anthropogenic factors on riverine ecosystems and emphasize the importance of managing human impacts to preserve microbial biodiversity and water quality.