In the framework of the WFD 2000/60/EC intercalibration process the updated versions of the EEI and R-MaQI, proposed by Italy and Greece for the transitional waters, have been applied to the macrophytes of the Venice lagoon to test their comparability and relationships with the pressure gradients. Submerged macrophytes were collected during spring 2007 at 60 sites spread within the lagoon. At each site, five random samples were collected and the total coverage of benthic macrophytes in the field was estimated based on a number of tests of the bottom. To assess seagrass epiphytes, five shoots were collected for each replicate. Physico-chemical data were collected in the water column at 14 sites selected to reflect the main hydro-geomorphological and trophic gradients of the lagoon. The analyses performed indicated that the two metrics appeared to be weakly intercalibrated and only the 30% of the sampling sites displayed the same quality class. The main differences fell into the Moderate and Low classes and the two indices provided discordant results in the intermediate and confined areas of the lagoon. In contrast, the two indices showed good affinity in the marine areas of inlets, which are characterised by seagrass meadows and late-successional macroalgae. Similar results were evidenced also in a redundancy analysis by the different relationships between quality classes and the physico-chemical gradients. The main reason for this seems to be the heterogeneity of species–environment relationships inside the groups of species on which the indices are based. Critical aspects of methodological differences and applicability of the macrophyte indices proposed by Italy and Greece for the transitional waters of the Mediterranean eco-region are discussed.