The dominant Mediterranean seagrass Posidonia oceanica inhabits sublittoral environments with very stable salinity regimes; the species is considered highly sensitive to even moderate increases in salinity caused by hypersaline effluents (brine) from desalination plants. We analysed the effect of salinity increase on seagrass vitality and survival by means of an in situ mesocosm experiment. To this end, we used the brine (70–75 psu) produced by a pilot desalination plant, which was diluted with seawater to obtain brine solutions of 1 psu (high salinity increase, HS) and 2.5 psu (very high salinity increase, VS) over the mean natural salinity (37.5±0.16 psu) and then interspersed a set of experimental units in a nearby P. oceanica meadow. At the end of the experimental period (3 months), these treatments had produced differential effects on all seagrass descriptors, i.e., intense and significant in the VS experimental units and more modest (or even negligible) in the HS ones. Seagrass meadow declined through a significant decrease in shoot density in both HS (12.4±3.4%, mean±SE) and VS (18.5±3.05%, mean±SE) in comparison to control experimental units (<5%). Surviving shoots had reduced size and lower leaf growth rate than control plants, but more generally a reduction in the photosynthetic leaf surface caused by the increase in the proportion of necrotic leaf area (up to 60% in VS treatments). Non-structural carbohydrate (soluble and reserve) concentrations decreased in rhizomes, probably in relation to physiological osmoregulation processes. All these responses support the hypothesis that the threshold salinity tolerance of P. oceanica is very close to the upper limit of its natural salinity range (ca. 38 psu).