Biomonitoring of fish fauna using environmental DNA methods (eDNA; sampling of shed DNA in water and detection of fish species via molecular techniques) could serve as a complementary technique to conventional, invasive, costly and time-consuming methods. Integration of eDNA metabarcoding (investigating the composition of entire fish communities) into existing regulatory monitoring frameworks, requires further insights and the development of practical guidelines. Remaining challenges include critical aspects of sampling and quantification of eDNA in lotic waters, as well as potential technical bias in eDNA detection and abundance quantification, spatial and temporal shifts in eDNA patterns, and assessments of ecological status. We will investigate the potential of eDNA methods for quality scoring of ecosystem health in rivers using the European Water Framework Directive and its implementation in Flanders as a practical starting point. By means of a large-scale sampling campaign, eDNA sampling will be compared to traditional electrofishing (TEF) to provide insights on spatio-temporal patterns of eDNA abundance in natural systems. Experimental validation and calibration of absolute measures for community abundance, optimization of metabarcoding workflow, as well as the transformation of eDNA metabarcoding measurements into discrete indices for biotic integrity, will bring about practical guidelines on the use of eDNA metabarcoding as a biomonitoring tool for ecosystem health.