Microalgae have a wide range of applications in biomass production and the production of bioactive compounds. The main research objectives were to determine the effect of different concentrations of inorganic selenium (sodium selenite) on the growth rate, photosynthetic activity, oxidative stress levels, and neutral lipid accumulation in the marine microalga D. tertiolecta, investigate the effect of selenite in the nutrient medium on lipid accumulation during one-stage and two-stage microalgae cultivation, and examine the feasibility of increasing microalgal growth and lipid synthesis in a laboratory photobioreactor with the addition of selenite. The highest values of optical density (OD₆₈₀=0.67) and cell density (43.11×10⁵ cells mLˉ¹) were achieved on the 10th day of two-stage cultivation in a laboratory photobioreactor in a medium enriched with 20.00 mg Lˉ¹ of selenite. Exposure of the microalga D. tertiolecta to elevated concentrations of selenite (20.00 and 40.00 mg Lˉ¹) resulted in an increased concentration of malondialdehyde (MDA) and other thiobarbituric acid reactive substances (TBARS), indicating lipid peroxidation in microalgal cells. In both small (100 mL) and large-scale (2 L) cultivation, the highest increase in neutral lipid content was recorded on the 14th day of two-stage cultivation in a microalgal culture containing 20.00 mg Lˉ¹ of selenite. During cultivation in the laboratory photobioreactor, the highest neutral lipid content was recorded on the 11th day of cultivation with 20.00 mg L-1 of selenite, and it was 27.67 times higher than the highest lipid content obtained during microalgal cultivation on a smaller scale (100 mL). Comparison of the fatty acid composition showed a significant increase in the proportion of palmitic (C16:0) and oleic acid (C18:1 cis 9), which is favorable for biodiesel production. Additionally, the fatty acid composition analysis revealed the presence of various fatty acids important for the production of polyunsaturated fatty acids (palmitoleic (C16:1 cis 9), linoleaidic (C18:2 trans 9, 12), linolenic (C18:2 cis 9, 12), and γ-linolenic acid (C18:3 cis 6, 9, 12)). Comparing the calculated values of four essential physical properties of biodiesel (cetane number (Φb), kinematic viscosity (ηb), density (ρb), and higher heating value (δb)) with standard values (ASTM D6751 and EN 14214), it was concluded that biodiesel produced by transesterification of microalgal biomass lipids grown at different concentrations of selenite meets all the requirements for biodiesel production standards. Adding selenite to the nutrient medium of the microalga D. tertiolecta represents a potential strategy to increase the yield of neutral lipids and improve the potential use of D. tertiolecta microalgal lipids for biodiesel production.