The current study is aimed at understanding the effect of two different concentrations of nitrate (NaNO3) i.e., 2.94 mM (1X) and 8.82 mM (3X) on the productivity of Scenedesmus sp. in terms photosynthetic efficiency, growth, biomass and protein/lipid synthesis. The experiments were conducted by growing the microalgae in mixotrophic mode with a fixed dissolved organic carbon (110 mM). Chlorophyll a fluorescence fast kinetics parameter such as F-V/F-M, F-M/F-O, P-i Abs, TRo/RC and ABS/RC depicted an improved PSII efficiency in 3X conditions. Higher nitrate concentration in BBM medium favored better assimilation of chlorophyll pigments, carbohydrates (160 mg/g), proteins (524 mg/g) and total lipids along with higher biomass (11.4 g/L). The microalgae cell growth, biomass and biochemical composition are significantly influenced by excess nitrates supplementation in the growth medium.