The microorganisms in sediments play a crucial role in biogeochemical cycle processes, and numerous studies have shown that microbial community is closely related to environmental factors. However, the usability of sediment microorganisms to evaluate the eco-environment quality of rural rivers has not been adequately explored. This study investigated the distribution characteristics and response of sediment microorganisms to environmental parameters and benthic organisms. Based on the environmental parameters and benthic community indices, the 12 stations were divided into high-polluted group A, moderate-polluted group B and low-polluted group C. Station DG01 and DG02 in group A had the highest level of As and Ni pollution and nutrient concentration, and DG09 in group A had the lowest benthic diversity. Correspondingly, group A had the lowest abundance of Proteobacteria, which has a higher requirement for the environment than Planctomycetes. Group B had the highest sulfide level (97.45 mg/kg), and bacteria (Thiobacillus, Sulfurisoma and Sulfuritalea) with genes involved in sulfur cycling were more enriched in this group. Group C had the lowest level of total nitrogen (243.36 mg/kg), and Rhodanobacteraceae in Xanthomonadales might be a key bioindicator for low nitrogen. In addition, Chlorophyta was found to be more susceptible to heavy metals, and moreover co-occurrence networks showed that microeukaryotes were more sensitive to heavy metal pollution compared to benthic animals and prokaryotes. Therefore, this study suggested that benthic microorganisms especially microeukaryotes could be used as good indicators for evaluating the eco-environmental quality of rural rivers.