<p>Wetlands have been drained extensively for productive land use, transforming the biodiversity of whole landscapes. Such transformation primarily affects the huge biodiversity across the terrestrial and aquatic environments that is difficult to observe directly, especially in the case of microorganisms. We explored whether environmental DNA (eDNA) from the flowing water could serve as a data source for characterizing the level of biological degradation of drained and managed forest-wetland landscapes. We took spatially and seasonally replicated samples from nine hydrologically monitored outflows at small drained catchments in Estonia in order to understand the variation in their eDNA-based diversity. Using PacBio long-read sequencing, we detected a large taxonomic diversity of eukaryotes (approx. 6000 operational taxonomic units (OTUs)), which was spatially and seasonally structured, but also highly variable within individual ditches. Even in fungi (the best-represented taxon group), the OTU accumulation curves did not level off despite high volumes of filtered water; however, many interesting species records were obtained (particularly on pathogenic microorganisms). We conclude that eDNA can provide valuable insights into the biodiversity of hydrologically drained areas, but our results indicate high heterogeneity among samples (apparently due to both actual assemblage differences and sampling errors) as a major problem for standard environmental assessment. Combining eDNA methods with other ecological assessment techniques is a priority for further research in these systems.</p>