The microorganisms in sediments play a significant role in Arsenic (As) migration in groundwater systems. However, the impact mechanisms of microbial community structure on As release and enrichment are not completely clear. In this study, the community structure and characteristics of microorganisms in sediments of the Kuitun River Basin were first investigated through field investigation, high-throughput sequencing, and microbial analysis. The obtained results showed that Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes were the dominant phyla in the sediments, accounting for 30.23%–87.87%, 3.280%–65.22%, 1.71%–14.37%, and 0.46%–16.67%, respectively. Whereas, Arthrobacter , Acinetobacter , Pseudomonas , and Hydrogenophaga were the main genera in the collected sediments from the Kuitun River Basin, accounting for 1.81%–60.13%, 0.70%–77.24%, 0.21%–35.5%, and 0.38%–26.27%, respectively. Arthrobacter can increase the As contents in the sediments. In contrast, Acinetobacter can both inhibit and promote the release of As from the sediments, while Pseudomonas and Hydrogenophaga can only inhibit the release of As from the sediments. The Variance Inflation Factor (VIF) suggested that Ca, Mg, Mn, Cu, and As were highly correlated with each other. The distance-based redundancy analysis (Db-RDA) analysis demonstrated significant influences of the sediment chemical properties on the microbial activity and community structure in the sediments, according to the following order: Ca > Cu > Mn > Mg > As. Ca ²⁺ and Mn ²⁺ in the environment can influence the growth and metabolism of microorganisms, thus affecting the redox environment and As release from sediments. This study confirmed the interaction that may exist between microorganisms and As. Moreover, this study not only confirmed the interaction between microorganisms and As, but also provided a comprehensive understanding of the effects of the microbial community on the chemical cycle of the groundwater system in the Kuitun River basin. The analysis of the influences of the microbial community on sediment As provided further insights into As release from sediments and As enrichment in groundwater in the study area.