Benthic macrophytes from 19 sites within the Marano-Grado Lagoon were sampled in July 2007 in order to update the available information on the flora and vegetation and to assess the Ecological Status (ES) within the European Water Framework Directive (WFD). Data on macrophytes were analysed using two phytobenthic indices (EEI and R-MaQI) recently set up to evaluate the ecological status of transitional environments in the Mediterranean eco-region. Notwithstanding the extension (c. 160 km2) that places this lagoon as the second largest Italian transitional environment, ecological studies are relatively few. The present research revealed a relatively poor flora, mainly characterized by the dominance of low-diversity settlements of Ulvaceae. Moreover, the absence of intensive aquaculture activities and commercial big harbours, which account for the new species introductions recorded in other lagoons, limits the presence of non-autochthonous species. The comparison with previous data (Curiel et al. 1998) highlighted the reduction of macrophyte coverage and biomass, especially Ulvaceae stands, and an increase in species richness. In particular, there was evidence of a worsening of the area north of the Grado bridge. This area, which in the past was colonized by widespread angiosperm coverage, at present is almost lacking in vegetation. According to these observations, by applying both the phytobenthic indices available in the literature for the transitional environments, the Marano-Grado Lagoon showed a ‘Good–High’ quality in the central part of the basin and near the lagoon inlets and a ‘Poor–Bad’ quality in the northern and southern areas. The evaluation of some physico-chemical parameters, coupled with the distribution of the angiosperms, also allowed a first delineation of the main lagoon water bodies.