Biodiversity within riverine systems is crucial for the ecosystem’s health and function, yet many of these ecosystems are being disturbed through increased human impacts, threatening the biodiversity within. While the general patterns of decreased biodiversity within riverine systems are well documented, little is known about how biodiversity across multiple taxonomic groups responds to land uses in the surrounding watershed. Environmental DNA (eDNA) can be used to efficiently measure the biodiversity and taxonomic composition of multiple species groups through sampling traces of DNA shed by organisms into the surrounding environment. In this study, we used eDNA metabarcoding to assess alpha and beta diversity across taxonomic groups at sites spanning a gradient of agricultural land use along the Mackinaw River watershed in central Illinois. To assess land use, we created a GIS map with 5-km buffers around each eDNA sampling site and applied a land use raster map to determine agricultural impact. We assessed biodiversity using eDNA metabarcoding with 12S and 18S primers to target fishes and eukaryotes, respectively. Our results revealed that alpha diversity of fishes was significantly higher in slightly and moderately disturbed sites, while eukaryotes were the most diverse in highly disturbed sites. Beta diversity among sites was high for both species groups. These findings demonstrate how land use in the surrounding watershed can impact the biodiversity of aquatic organisms across different taxonomic groups, providing important insights into potential conservation strategies to sustain the biodiversity of freshwater systems.