Seagrasses, which are considered among the most ecologically valuable and endangered coastal ecosystems, have a narrowly limited distribution in the south-east Pacific, where Zostera chilensis is the only remaining relict. Due to water scarcity, desalination industry has grown in the last decades in the central-north coasts of Chile, which may be relevant to address in terms of potential impacts on benthic communities due to their associated high-salinity brine discharges to subtidal ecosystems. In this work, we assessed ecophysiological and cellular responses to desalination-extrapolable hypersalinity conditions on Z. chilensis. Mesocosms experiments were performed for 10 days, where plants were exposed to 3 different salinity treatments: 34 psu (control), 37 psu and 40 psu. Photosynthetic performance, H2O2 accumulation, and ascorbate content (reduced and oxidized) were measured, as well as relative gene expression of enzymes related to osmotic regulation and oxidative stress; these, at 1, 3, 6 and 10 days. Z. chilensis showed a decrease in photosynthetic parameters such as electron transport rate (ETRmax) and saturation irradiance (EkETR) under hypersalinity treatments, while non-photochemical quenching (NPQmax) presented an initial increment and a subsequent decline at 40 psu. H2O2 levels increased with hypersalinity, while ascorbate and dehydroascorbate only increased under 37 psu, although decreased along the experimental period. Increased salinities also triggered the expression of genes related to ion transport and osmolyte syntheses, but salinity-dependent up-regulated genes were mostly those related to the reactive oxygen species metabolism. The relict seagrass Z. chilensis has shown to withstand increased salinities that may be extrapolable to desalination effects in the short-term. As the latter is not fully clear in the long-term, and considering the restricted distribution and ecological importance, direct brine discharges to Z. chilensis meadows may not be recommended.