Microalgal biomass is characterized by high protein, carbohydrates, and lipids concentrations. However, their qualitative and quantitative compositions depend not only on the cultivated species but also on the cultivation conditions. Focusing on the microalgae’s ability to accumulate significant fatty acids (FAs) amounts, they can be valorized either as dietary supplements or for biofuel production, depending on the accumulated biomolecules. In this study, a local isolate (Nephroselmissp.) was precultured under autotrophic conditions, while the Box–Behnken experimental design followed using the parameters of nitrogen (0–250 mg/L), salinity (30–70 ppt) and illuminance (40–260 μmol m−2s−1) to evaluate the accumulated biomolecules, with an emphasis on the amount of FAs and its profile. Regardless of the cultivation conditions, the FAs of C14:0, C16:0, and C18:0 were found in all samples (up to 8%w/win total), while the unsaturated C16:1 and C18:1 were also characterized by their high accumulations. Additionally, the polyunsaturated FAs, including the valuable C20:5n3 (EPA), had accumulated when the nitrogen concentration was sufficient, and the salinity levels remained low (30 ppt). Specifically, EPA approached 30% of the total FAs. Therefore,Nephroselmissp. could be considered as an alternative EPA source compared to the already-known species used in food supplementation.