This study investigates the benthic invertebrate community in the Venice Lagoon using environmental DNA (eDNA) metabarcoding based on superficial water samples. Our objective is to understand if, in a shallow lagoon system, sampling at the surface can provide information on benthic biodiversity, allowing us to establish a baseline for future assessments and to monitor the community's seasonal and spatial variation. eDNA was collected from surface water samples at two sites during nine sampling campaigns from November 2018 to October 2019, and metabarcoding was performed using an available primer pair targeting 16S mitochondrial rDNA of echinoderms, never tested empirically before. Analyses revealed 80 taxonomic units, five not assigned at the species level, belonging not only to the phylum Echinodermata but predominantly assigned to Mollusca, with the majority of the identified species (60 out of 75) representing benthic invertebrates. Several species known to be invasive were detected, some previously recorded in the Venice Lagoon environment and others detected for the first time. Significant spatial differences in species composition were observed between the northern and the southern site of the lagoon. Temporal variation of the benthic community was also observed, mainly due to the distinctiveness of autumn samples, highlighting the dynamic nature of the Venice Lagoon ecosystem. Our results confirm the utility of eDNA for ongoing biodiversity monitoring and management and show that eDNA isolated from superficial water can provide information on the benthic invertebrate community, which might be particularly useful for biodiversity assessment in lagoons, ports, and areas characterized by limited or interdicted access.