Ecotoxicological risk of ZnO nanoparticles at environmental levels is a key knowledge gap for predicting how freshwater ecosystems will respond to nanoparticle pollution. A microcosm experiment was conducted to explore the chronic effects of ZnO nanoparticle at environmental concentrations (30, 300, 3000 ng L) on aquatic fungi associated with the decomposing process of poplar leaf litter (45 days). ZnO nanoparticles led to 9-33% increases in fungal biomass after acute exposure (5 days), but 33-50% decreases after chronic exposure (45 days), indicating that the hormetic effect of ZnO nanoparticles at the environmental level may occur during acute exposure. Besides, ZnO nanoparticles had negative effects on microbial enzyme activity, especially on day 10, when the activities of N-acetylglucosaminidase, glycine-aminopeptidase, aryl-sulfatase, polyphenol oxidase, and peroxidase were significantly inhibited. After chronic exposure, the fungal community structure was significantly impacted by ZnO nanoparticles at 300 ng L due to the reduced proportion of Anguillospora, which eventually caused a significant decrease in litter decomposition rate. Therefore, ZnO nanoparticles may pose ecotoxicological effects on aquatic fungi even at a very low concentration and eventually negatively affect freshwater functioning.