This study investigated the impact of culture medium salinity (5–50 PSU) on the growth and maximum photochemical yield of photosystem II (Fv/Fm) and the composition of carotenoids, fatty acids, and bioactive substances in three marine microalgae (Chrysochromulina rotalis, Amphidinium carterae, and Heterosigma akashiwo). The microalgae were photoautotrophically cultured in discontinuous mode in a single stage (S1) and a two-stage culture with salt shock (S2). A growth model was developed to link biomass productivity with salinity for each species. C. rotalis achieved a maximum biomass productivity (Pmax) of 15.85 ± 0.32 mg·L⁻¹·day⁻¹ in S1 and 16.12 ± 0.13 mg·L⁻¹·day⁻¹ in S2. The salt shock in S2 notably enhanced carotenoid production, particularly in C. rotalis and H. akashiwo, where fucoxanthin was the main carotenoid, while peridinin dominated in A. carterae. H. akashiwo also exhibited increased fatty acid productivity in S2. Salinity changes affected the proportions of saturated, monounsaturated, and polyunsaturated fatty acids in all three species. Additionally, hyposaline conditions boosted the production of haemolytic substances in A. carterae and C. rotalis.