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Chemical-based water quality assessments, while widely used, often fail to capture the cumulative and fluctuating impacts of nutrient pollution. Diatoms, as primary producers in freshwater systems, serve as valuable bioindicators due to their rapid response to environmental changes. However, traditional microscopy-based diatom identification is limited by observer variability, morphological ambiguity and potential underestimation of cryptic or damaged taxa. To address these issues, we implemented an environmental DNA (eDNA) metabarcoding approach to assess diatom communities and calculate nutrient sensitivity metrics. Our study analyzed biofilm eDNA samples from 209 sites across Korea, covering both pristine forested headwaters and nutrient-enriched streams near urban and agricultural areas. Using rbcL gene primers and high-throughput sequencing, we identified 329 diatoms in 60 genera. Diatom community composition displayed distinct patterns related to phosphorus concentrations. We employed Indicator Species Analysis (ISA) based on PO 4 -P groupings to derive species-specific sensitivity (S) and indicator (I) values, which were integrated into a Trophic Diatom Index (TDIe) for ecological health assessment. TDIe values significantly decreased with rising PO 4 -P levels, illustrating degraded ecological conditions. Compared to the microscope-based TDI (TDIm), the eDNA-derived TDIe demonstrated statistically significant correlations with PO 4 -P and total phosphorus (TP) concentrations ( r = −0.48 and −0.37, respectively), indicating enhanced sensitivity and environmental relevance. Notably, at several sites, TDIe detected ecological degradation not captured by TDIm, including a reclassification of some previously underestimated nutrient-tolerant or sensitive taxa. This study demonstrates that eDNA-based diatom assessments can overcome the major limitations of traditional microscopy by offering a scalable and objective method for evaluating ecological conditions. By detecting a broader range of taxa, including rare or damaged forms, this approach improves the resolution of bioassessment metrics. However, further standardization of regional barcode databases and methodological refinements are necessary for its broader application. Overall, eDNA metabarcoding provides a promising tool for supplementing conventional biomonitoring programs and advancing the accuracy of freshwater ecosystem health evaluations. • eDNA metabarcoding enhances diatom detection, surpassing traditional microscopy. • Trophic Diatom Index (TDIe) from eDNA correlates strongly with nutrient concentrations. • Biofilm-derived eDNA reveals nutrient-sensitive diatom species across diverse habitats. • eDNA-based diatom assessment detects ecological degradation missed by microscopy. • This study provides a scalable eDNA approach for freshwater bioassessment.