The Ria de Vigo (NW Spain) is a complex socio-ecological system where urban, industrial and aquaculture activities overlap, generating interactions that compromise ecosystem health. This study investigated the spatial distribution of putative eukaryotic pathogens near wastewater discharge points from the municipalities of Vigo, Cangas, and Redondela. A multimarker environmental DNA metabarcoding approach was employed, targeting the V4 and V9 regions of the 18S rRNA gene and the ITS-2 region for fungi in water and sediment samples. Eukaryotic diversity varied by location and genetic marker, with inner estuary sediments exhibiting the highest taxonomic richness, and mid-estuary zones showing the lowest. The V9 region revealed broader taxonomic coverage, including rare taxa, while the V4 region yielded higher read abundances. Putative pathogenic taxa were frequently detected in inner areas. Genetic material from human and zoonotic pathogens, aquaculture pathogens and harmful algae (Alexandrium minutum) were detected. The study highlights three components for effective eDNA-based pathogen monitoring: using multi-marker analysis, including sediment sampling, and incorporating landscape-scale variables. Together, these strategies enhance detection and management of biohazard in coastal environments.