Over the past century, Lake Garda has become a major hotspot for biological invasions, with nearly 50 non-indigenous species recorded to date and new introductions continuously occurring. While traditional monitoring methods typically detect these invaders only after they have successfully established, advanced molecular tools offer the potential for early-stage detection. In this context, lacustrine harbors represent critical entry points, yet they are frequently overlooked in standard biological inventories and dismissed as degraded, anthropogenically impacted areas. Thus, we implemented an environmental DNA metabarcoding approach, sampling water and sediment across eight major harbors around the lake to thoroughly characterize their biodiversity, track alien species presence, and unravel connectivity patterns. Preliminary molecular data, based on COI and 18S rRNA genes, successfully captured a remarkably rich biota encompassing protists, algae, and metazoans. Beyond documenting several alien and undocumented endemic species, this high-throughput sequencing approach unveiled broader catchment-to-lake connectivity. Notably, the detection of terrestrial species' DNA highlights how the lake acts as a sink for runoff and land-water interactions. Ultimately, integrating traditional and molecular approaches bridges the gap from organismal detection to ecosystem-wide monitoring, demonstrating that these heavily impacted artificial habitats actually host high biological diversity and provide key data on watershed-level dynamics.