Nonpoint source pollution (NPSP) from human production and life activities causes severe destruction in river basin environments. In this study, three types of sediment samples (A, NPSP tributary samples; B, non-NPSP mainstream samples; C, NPSP mainstream samples) were collected at the estuary of the NPSP tributary of the Jialing River. High-throughput sequencing of the fungal-speci c internal transcribed spacer (ITS) gene region was used to identify fungal taxa. The impact of NPSP on the aquatic environment of the Jialing River was revealed by analysing the community structure, community diversity and functions of sediment fungi. The results showed that the dominant phylum of sediment fungi was Rozellomycota, followed by Ascomycota, Chytridiomycota, Basidiomycota, Mortierellomycota and Zoopagomycota (relative abundance>1%). NPSP caused a signi cant increase in the relative abundances of Rozellomycota, Saccharomycetes, Microascales, Saccharomycetales, Branch02 and Branch03. In addition, it caused a signi cant decrease in the relative abundances of Chytridiomycota, Dothideomycetes, Capnodiales, Glomerellales, Xylariales and Chaetothyriales.Moreover, NPSP caused signi cant changes in the physicochemical properties of Jialing River sediments, such as pH and available nitrogen (AN), which signi cantly increased the species richness of fungi and caused signi cant changes in the fungal community β-diversity (P<0.05). pH, total phosphorus (TP) and AN were the main environmental factors affecting fungal communities in Jialing River sediments. The functions of sediment fungi mainly involved three types of nutrient metabolism (symbiotrophic, pathotrophic and saprotrophic) and 75 metabolic circulation pathways. NPSP signi cantly improved the NONOXIPENT-PWY, PENTOSE-P-PWY, and PWY-6837 metabolic circulation pathway functions (P<0.05) and inhibited the PWY-7118, PWY-5920, and PWY-6609 metabolic circulation pathway functions (P<0.05). Hence, NPSP causes changes in the community structure and functions of sediment fungi in Jialing River and destroys the stability of the Jialing River Basin ecosystem.