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Transitional environments have great ecological value and high productivity, and many species can benefit from their sheltered conditions and food resources. In this study, we performed for the first time a fish-targeted eDNA metabarcoding of the 12S rRNA gene at 16 sites broadly covering the different water bodies of the Marano and Grado Lagoon (northern Adriatic Sea) in two seasons. The eDNA was collected at the same time as the beach seine net, allowing a direct comparison of the two approaches. With eDNA we detected 34 species, covering all the functional guilds occurring in the lagoon. Species of regional interest, that uses the area as a nursery and feeding ground, and diadromous species, highlighting the ecological connectivity between freshwater and marine habitats, were found. While some species were constantly present (e.g. Atherina boyeri, Sparus aurata), others (Squalius cephalus, Platichthys flesus) were influenced by salinity (higher in Grado and lower in Marano), which was confirmed as the main ecological driver in this environment. The comparison with traditional methods, which identified 18 species (11 of which were detected with both approaches), showed that eDNA is very sensitive in detecting most of the biodiversity in the lagoon with a limited sampling effort. Few relevant species (Chelon saliens , Knipowitschia panizzae) lacked reference sequences, which need to be implemented in the databases. Our study represents a significant advance in the understanding of lagoon fish biodiversity and ecological dynamics and contributes to the improvement of management strategies in these ecologically sensitive habitats. • eDNA detected a great richness of fish species, across different functional groups • The molecular approach (eDNA) was more sensitive than the traditional one (seine net) • Reference databases still lack sequences of relevant lagoon fish species [ABSTRACT FROM AUTHOR]