The microalga, Dunaliella salina, is a treasure of fine chemicals, carotenoids, and lipids of industrial importance. The present investigation focused on the maximization of lipid content in the context of biofuel. Mixotrophic cultures growing in different combinations of sodium bicarbonate (NaHCO3) and glucose (Glu) or sodium acetate (Na-Ace) were monitored for essential growth parameters such as photosynthesis, dry biomass, and Chl a content. The maximum photosynthetic quantum yield (Fv/Fm) of all the screened mixotrophic cultures along with control ranged between 0.63 and 0.72, indicating the cells were in a good physiological state. The highest biomass productivity (33.46 ± 0.58 mg/L/d) was recorded in 1.0 mM NaHCO3 + 1.0 mM Glu-containing cultures. However, the highest lipid content (56% DCW) was recorded in 1.0 mM NaHCO3 + 0.5 mM Na-Ace-containing cultures. Interestingly, a 1.7-fold higher starch content (72.3 μg/mL) was recorded in 0.25 mM NaHCO3 + 0.5 mM Na-Ace over 1.0 mM NaHCO3 + 0.5 mM Na-Ace-containing cultures. Thus, data indicated that the higher concentration of NaHCO3 along with Na-Ace favoured lipid biosynthesis, while the lower concentration of NaHCO3 with Na-Ace favoured starch synthesis. Thus, substrate provision determined the desired products. Lipids obtained from D. salina at stage II (nitrogen-deficient condition) possessed ideal fuel properties according to international standards of EN 14214 and ASTM D6751. Therefore, the present work explored the threshold level of bicarbonate along with Na-Ace which was able to maintain good physiological status and produced higher lipids with better biodiesel properties. [ABSTRACT FROM AUTHOR]