Marine biodiversity monitoring in the Mediterranean’s increasingly threatened ecosystems is crucial for effective ecosystem conservation and management. Here, we leveraged the Mediterranean International Trawl Survey program (MEDITS) to implement eDNA sampling through the recently tested ‘metaprobe’ procedure and characterize fish assemblages in three separate areas off the Italian coasts: Northern Adriatic Sea (NoAS), Ligurian and Northern Tyrrhenian Sea (LNTS), and Sardinian Sea (SaS). By combining the information from two homologous mitochondrial 12S metabarcodes––i.e., Elas02 and Tele02 targeting elasmobranchs and teleosts, respectively––we identified 108 species, over 60% of which overlapped with those caught by the trawl net. We produced an accurate reconstruction of fish community composition of the examined sites, reflecting differences in species assemblages linked with both geographic area and depth range. Metaprobe eDNA data consistently returned a biodiversity ‘bonus’ mostly consisting of pelagic taxa not captured through bottom trawl surveys, including rare and endangered taxa (e.g., elasmobranchs). Overall, the spatial characterisation of the assemblages across the surveyed areas was better delineated and more robust using eDNA metabarcoding than trawl data. Our results support the operationalisation of the metaprobe as a simple, inexpensive, versatile sampling tool, in association with pre-existing ship surveys, to overcome many of the limitations of marine data collection and strengthen marine management.