Environmental DNA (eDNA) metabarcoding offers an efficient approach for assessing biodiversity across space and time. A key benefit of eDNA approaches is the ability to target multiple taxonomic groups using different genetic markers, providing a more comprehensive view of biodiversity across trophic levels within ecosystems. Here, we applied a multi-marker eDNA framework to characterize biodiversity patterns across a gradient of land use in an agriculturally dominated watershed. To do so, we analyzed three loci targeting different taxonomic groups: 18S rRNA (broad eukaryotic diversity), COI (macroinvertebrates), and 12S rRNA (fish). We compared within- (α-diversity) and among-site (β-diversity) biodiversity patterns across sites spanning a gradient of land use intensity, as indicated by the percent agriculture and urban cover within the upstream catchment of each sampling site. We found divergent responses among target species groups, with sites with higher land use intensity exhibiting higher overall eukaryote diversity, while fish and macroinvertebrate diversity was lower at more impacted sites. These contrasting results suggest that land use impacts may reduce diversity in traditional bioindicator species groups while increasing overall eukaryotic diversity, potentially due to shifts toward disturbance-tolerant microeukaryotic taxa. Despite these differences in α-diversity, all species groups showed high β-diversity across sites, indicating strong turnover in community composition across the land use gradient. By combining fish and macroinvertebrate metabarcoding with broad eukaryotic diversity metrics, this approach provides a more complete picture of how watershed land use impacts freshwater biodiversity across multiple trophic levels.