Salinity fluctuations promote adverse effects on the immunity of marine bivalves; however, sometimes experimental data are controversial. In the present work, we evaluated in vivo effects of short-time salinity changes on hemocyte functions of Mediterranean mussel, Mytilus galloprovincialis. Mussels were acclimated to hyposaline (6, 10, 14) and hypersaline (24,30) conditions and exposed to altered salinity for 2 days. Then hemolymph cellular composition, hemocyte immune (intracellular reactive oxygen species (ROS) production, phagocytic activity), and functional parameters (morphology, mitochondrial membrane potential) were measured using flow cytometry and fluorescent microscopy. Salinity fluctuations led to an increase of ROS production by hemocytes in all experimental groups. Increased ROS levels were accompanied with elevated mitochondrial membrane potential for all treatments except salinity 30, where the parameter decreased significantly. Salinity stress (all experimental groups) inhibited phagocytosis in agranulocytes, whereas stimulated phagocytic activity of granulocytes at salinity 14 and 24 indicating that moderate salinity fluctuations may cause stimulating effect on immunocytes of bivalves. Hypersaline and hyposaline treatments did not affect hemocyte morphology. These results indicate that hemocytes likely possess physiological mechanisms that restore initial cellular volume following hypoosmotic swelling/hyperosmotic shrinkage. Salinity stress affects hemocyte functionality of bivalves with varying intensity.