Optimization of the fatty acid profile in microalgae is one of the key strategies for obtaining valuable products and sustainable biofuels. Light intensity and light regimes exert an impact on the growth and metabolic process in microalgae. The objective of the present investigations was to assess the effect of light intensity and continuous light vs. photoperiod conditions on the growth and changes in the biomass composition in Eustigmatos magnus, with a focus on bioactive molecules such as lipids and fatty acids. The highest daily productivity of Eustigmatos magnus biomass and lipid yields were detected at continuous illumination and at the highest intensity of light. The results show that the content and composition of fatty acids was influenced by the culture conditions. The biomass of Eustigmatos magnus contained the highest concentrations of polyunsaturated fatty acids in the pphotoperiod conditions with the highest light intensity. This study shows that Eustigmatos magnus has a capacity for the accumulation of palmitoleic acid. A high intensity of continuous light improves the profile of fatty acids in Eustigmatos magnus, which can be suitable for biodiesel applications. At the high intensity of continuous light, Eustigmatos magnus lipids are characterized by high content of oleic acids and low content of saturated and monounsaturated acids.