High‑arsenic (As) groundwater in geothermal aquifers poses a serious threat to public health. Assembly processes governing groundwater microbial community related to As biotransformation are still unexplored in geothermal groundwater across different aquifers. To fill this gap, groundwater microorganisms, community assembly processes, and microbially metabolic coupling of carbon (C), nitrogen (N), phosphorus (P), sulfur (S), and arsenic (As) were investigated in unconfined and confined groundwater in the thermal reservoirs of the Guide Basin. The difference in groundwater hydrogeochemicals led to the heterogeneity of the microbial community and microbially mediated C, N, P, S, and As cycling between unconfined and confined groundwater. Higher temperature and As concentrations, low nutrient supply, and reduced conditions in confined groundwater supported stronger interspecific coexistence and environmental selection, thus promoting the proliferation of As-resistant microorganisms (ARMs) and simplifying the community assemblage. Abundant available nutrient supply and oxidizing conditions supported an increased species diversity and metabolic functionality in unconfined groundwater. S oxidizers, C fixation, and C degradation bacteria potentially contributed to the decreased As concentrations in unconfined groundwater. However, ARMs, ammonification, and anaerobic ammonia-oxidizing bacteria potentially caused As mobilization in confined groundwater. Overall, our results give a comprehensive insight into the interaction between As and microorganisms in geothermal groundwater.