Microorganisms are sensitive to changes in the external environment and are often used as indicators to monitor and indicate water quality. We decided to investigate the characteristics of microbial community structure in the sediment of the Guishui River and understand the impact of environmental factors on microbial community distribution, to ensure that water quality requirements are met for hosting the World Horticultural Exhibition and Winter Olympic Games in Yanqing District. Water and sediment samples from 12 different sections of the Guishui River were collected to analyze the physical and chemical properties of water and study the microbial community structure characteristics of sediment. It was found that the water quality of chemical oxygen demand (COD), ammonia nitrogen (NH₄⁺-N), and total nitrogen (TN), were below the standard in the middle and lower reaches of Guishui River, and the total nitrogen content of the sediment had significant positive correlation with that of the overlying water (P=0.914). The microbial community structure was investigated using MiSeq high-throughput sequencing. The bacterial species detected in these samples covered 70 phyla, 228 classes, and 1168 genera, and it was found that Proteobacteria, Bacteroidetes, Acidobacteria, Actinobacteria, Chloroflexi, Firmicutes, Cyanobacteria, Chlorobi, Verrucomicrobia, and Nitrospirae, were the main bacteria in the sediment, accounting for more than 84% in each sample, and the phylum Proteobacteria was most abundant, accounting for 45.3% to 69.1% of the total sample. The dominant species varied in different cross-sections of the samples. The abundance and diversity of microbial communities in the sediments of Guishui River were relatively high, but the Ace richness index and the Shannon diversity index (2673 and 6.56, respectively) of the D7 point in the Shiyuan section were lower than at other points. The result of redundancy analysis showed that water dissolved ammonia nitrogen and temperature were the main environmental factors influencing the microbial communities (F=2.92,P=0.038 and F=2.81,P=0.014, respectively). The dominant bacteria in the sediments were of the genus Dechloromonas and Thiobacillus. Their abundance is positively correlated with the content of ammonia nitrogen and water temperature, and negatively correlated with the content of dissolved oxygen. The research results provide data support and theoretical guidance for ecological environment protection and water quality management of Guishui River.