Most of the studies on biological and ecological effects associated with brine discharge into the marine environment are related to the operation of desalination plants, for the production of freshwater. In this study we analysed the biological effects of a brine effluent from a completely different source, produced from the lixiviation of rock salt caves, for the creation of natural gas storage caverns. Lethal and sub-lethal endpoints following exposure to the brine were studied in a range of macrofauna species characteristic of the soft and hard bottom habitats in the vicinity of the discharge area, namely the isopod Eurydice pulchra, the annelids Sabellaria alveolata and Ophelia radiata, the sea-urchin Paracentrotus lividus and the bivalve Mytilus galloprovincialis. In a first series of experiments, brine, with salinity above 300, was diluted in distilled water to a salinity value close to that of the seawater in the discharge area (salinity 36) and, surprisingly, none of the exposed species was able to survive or develop into viable larvae. A second series of experiments exposed the species to brine diluted with seawater, simulating more realistic discharge circumstances. All the tested species at all the measured endpoints (adult survival, larval abnormal development, sperm fertilization success) showed negative biological effects in brine solutes always at a lower salinity than that of a salinity control obtained with concentrated seawater. The sub-lethal experiments with larval development of P. lividus, S. alveolata and M. galloprovincialis, and the fertilization success of P. lividus gave EC 50 values for the brine solute with salinity in the range of 40.9–43.5, whereas the EC 50 values for the concentrated seawater were in the range of salinity 44.2–49.0. It is hypothesised that differences in the ionic composition of the brine cause the inability of the species to tolerate the exposure to brine.