The aim of this study was to develop and validate a suite of sensitive responses (biomarkers) for monitoring conservation status and human impact in Posidonia oceanica meadows. Analytical methods were developed for NADPH cytochrome c reductase, ethoxicoumarin-o-deetilase (ECOD), guaiacol peroxidase (GPOX) and superoxide dismutase (SOD) assays. Western blot and a preliminary proteomic approach using 2-DE were also proposed as biomarkers. These techniques were initially tested on samples of posidonia experimentally exposed to various contaminants. Once validated, this approach was applied to Posidonia in a field study. Specimens of the seagrass were collected at four sites with potential different human impact along northern Tyrrenian coast. The results showed that reductase activity was significally induced in the various sampling areas compared to the reference site. GPOX and SOD showed a similar trend; the highest activities were measured in samples collected in front of a chlor-alkali plant and close to a river estuary. Analysis of residues (heavy metals, PAHs and OCs) measured on leaves showed differences between sites. A significant correlation was found between levels of Hg and GPOX and levels of Cr, Al and As and reductase activity. These results validated the potential use of the biomarkers approach on Posidonia for the assessment of ecotoxicological impact on the coastal environment.