This study investigated the effects of elevated salinity on the growth, morphology, and biochemical composition of two freshwater microalgae species: Chlorella thermophila (a green alga) and Anabaena variabilis (a cyanobacterium). The goal was to understand their adaptive mechanisms under saline stress and evaluate their potential for biofuel production. These species were chosen for their ecological significance and contrasting cellular structures (unicellular versus filamentous), which provide complementary insights into salinity tolerance among freshwater microalgae. Cultures were maintained in both standard BG11 medium and artificial seawater medium (SWM) under controlled light intensity (200 μmol photons m-2 s-1) and pH (7.5). Over a cultivation period of 10-20 days, we quantified key parameters, including cell size, volume, chlorophyll a, protein, lipid content, and fatty acid profiles, using microscopy (ImageJ), spectrophotometry, FTIR, and GC-MS analysis. Both species showed increased cell volume and lipid accumulation in SWM, with C. thermophila experiencing a dramatic volume increase from 5.83 μm3 to 54.76 μm3) and a 6.8% rise in lipid productivity. Fatty acid profiling identified distinct fatty acids, such as palmitoleic and pantetheic acids, in C. thermophila cultivated in SWM. These specific fatty acids may indicate adaptive strategies for osmoregulation or metabolic shifts toward energy storage under salinity-induced stress. These findings highlight the potential of salinity-driven modulation of microalgal metabolism to enhance biofuel precursors, offering a sustainable approach for biomass production in saline environments with reduced reliance on freshwater.