Lagoon environments, like all the marine-coastal areas, offer a wide variety of ecosystem services, but at the same time are affected by pressing human activities that lead to deterioration of the environmental quality, loss of biodiversity, habitat destruction and pollution. Since the well-being of population and local economy depend on the environmental status of these ecosystems, it is essential to adopt long-term management tools to achieve the Good Environmental Status sensu European Marine Strategy Framework Directive and Water Framework Directive. A Nature 2000 site (Lesina lagoon, south Italy) was assessed within a project aimed at protecting and restoring biodiversity and lagoon habitat through integrated monitoring, suitable management, and good practices. Here we provide an assessment of the lagoon integrity based on a multi-metrics approach, highlighting match/mismatch among environmental quality indicators and microplastics (MP) pollution. Some environmental quality indices based on ecosystem components as vegetation, macroinvertebrates and water trophic variables were applied together with an accurate evaluation of MPs abundance, distribution, and composition to assess the ecological status of Lesina lagoon before and after cleaning actions with litter removal. Overall, all the ecological descriptors outlined a clear lagoon spatial gradient, with a western saltier and organic-enriched area characterized by the absence of vegetation, macrozoobenthos with lower diversity and richness and high MPs occurrence. The focus on macrozoobenthos, considered as a key component of the lagoon ecosystem, identified much more sites in “poor” status than the other indicators here considered. Moreover, it was found a negative relationship between the Multivariate Marine Biotic Index and MPs items in sediment, suggesting that MP pollution has a negative impact on macrobenthic fauna, concurring to the deterioration of the benthic ecological status.