Coastal lagoons are considered important habitats both for ecological functions and biodiversity worldwide. Thus, they provide relevant ecosystem services and valuable natural resources. However, coastal lagoons are highly susceptible to anthropogenic pressures that can cause biodiversity losses and require specific biomonitoring programs as well as management measures. In this research, we applied environmental DNA (eDNA) metabarcoding to investigate the biodiversity of a poorly known Mediterranean lagoon included in the European Natura 2000 Network. We used the cytochrome oxidase I (COI) gene marker to capture the entire biodiversity of this highly diversified aquatic coastal environment. With a low sampling effort and rapid laboratory practices, a large amount of valuable biodiversity data was generated and analyzed. Interestingly, this straightforward and broad molecular surveying of biodiversity unveiled a wide variety of taxonomic groups, such as benthic macroinvertebrates, zooplankton, phytoplankton, and macroalgae, which are frequently used as ecological indicators. We were able to detect species that were previously morphologically identified, as well as species never identified before. This research underlines the validity of eDNA metabarcoding in assessing the biodiversity in a poorly known and protected Mediterranean lagoon ecosystem, as well as in identifying the early warnings of environmental stressors. Finally, the research highlights the need to investigate multiple target genes and primers set for a larger analysis of specific species.