Understanding the interactions among flow-sediment, microorganisms, and biogeochemical cycles is crucial for comprehending the ecological response mechanisms of dams and water diversion. This study focused on the spatial patterns of carbon, nitrogen, phosphorus, and sulfur (CNPS) cycle functional genes in the water resource for the middle route of the South-to-North Water Diversion Project in China, specifically the Danjiangkou Reservoir (comprising the Han and Dan reservoirs). The investigation incorporated sediment physicochemical properties and microplastic pollution. Numerous microbial species were identified, revealing that microbial communities demonstrated sensitivity to changes in sedimentary mud content. The communities exhibited greater β diversity due to finer sediment particles in the Han Reservoir (HR), whereas in the Dan Reservoir (DR), despite having higher sediment nutrient content and MPs pollution, did not display this pattern. Regarding the composition and structure of microbial communities, the study highlighted that sediment N and P content had a more significant influence compared to particle size and MPs. The quantitative microbial element cycling (QMEC) results confirmed the presence of extensive chemolithotrophic microbes and strong nitrogen cycle activity stemming from long-term water storage and diversion operations. The denitrification intensity in the HR surpassed that of the DR. Notably, near the pre-dam area, biological nitrogen fixation, phosphorus removal, and sulfur reduction exhibited noticeable increases. Dam construction refined sediment, fostering the growth of different biogeochemical cycling bacteria and increasing the abundance of CNPS cycling genes. Furthermore, the presence of MPs exhibited a positive correlation with S cycling genes and a negative correlation with C and N cycling genes. These findings suggest that variations in flow-sediment dynamics and MPs pollution have significant impact the biogeochemical cycle of the reservoir.