Seawater reverse osmosis (SWRO) desalination discharge brine-waste as part of the process with immediate (short-term) and chronic (long-term) impacts to marine coastal environment. Brine-waste is often denser than the receiving environment, therefore sinks and flows as a saline plume in adjacency to the sea floor. We suggest that the saline flow over the bottom may impose an osmotic stress, and alter benthic heterotrophic microbial diversity, activity and growth. In this study, we examined the short-term (48h) effects of different salinities on benthic heterotrophic bacteria attached to the sediment at the eastern Mediterranean coast. To this end, 12 cylinders were filled with sediment and incubated in full darkness with rising salinities ranging from 2% to 20% over the ambient levels. During the summer experiments, heterotrophic bacterial abundance has reduced by 60% at salinity levels >5% above the ambient concentration. Further, bacterial cell specific activity significantly increased following high salinity scenarios. Our experimental results provide the first scientifically-based data on the immediate effects of SWRO brine over benthic heterotrophic bacteria. However, we stress that long-term studies are imperative at the outfall of operating desalination facilities to determine the chronic effects of brine on benthic bacteria. Display omitted •Brine (salinity) discharge of 5% over the ambient reduces benthic bacteria abundance.•Short-term exposure to brine did not affect the diversity of benthic bacteria.•The metabolic response per cell was enhanced due short term exposure to SWRO brine.•The impact of a brine plume on the benthic bacteria may extend up to few hundreds of m2.