The effect of different light spectrum and CO2 concentrations on the growth and lipid content of Micractinium pusillum and Ourococcus multisporus was investigated. The highest biomass yields (2.9 and 2.6 g-dry cell weight l-1) were observed for M. pusillum and O. multisporus, respectively, at 5% CO2 with red light illumination. Red light spectrum with 5% CO2 supported the highest lipid contents (20% and 27%) and lipid productivity (32 and 36 mg l-1 d-1) for M. pusillum and O. multisporus, respectively. The highest fatty acid methyl esters content for both microalgal species was observed under red light spectrum and 5% CO2 conditions, with the oleic acid fraction ranging between 35% and 37%. This study showed that the red light spectrum and 5% CO2 were the optimum conditions for maximum growth, lipid content and lipid productivity of both microalgae species, which could be further exploited to establish a microalga-based biodiesel production strategy.