The focus of this study was to observe the growth of Chlorella sp., Nannochloris sp., and Botryococcus braunii under mixotrophic conditions (i.e., added glycerol) with the aim of increasing the growth of biomass and algae oil content. A significant growth of biomass was obtained when grown in glycerol rich environment comparing to autotrophic conditions. 5 g/L glycerol yielded the highest biomass concentration for these strains. Mixotrophic conditions improved both the growth of the microalgae and the accumulation of triacylglycerols (TAGs). The maximum amount of TAG in Botryococcus braunii was reached in the growth medium with 10 g/L glycerol and Chlorella sp., Nannochloris sp. with 2 g/L glycerol. The content of saturated fatty acids of Chlorella sp., Nannochloris sp., and Botryococcus braunii was found to be 34.94, 14.23 and 13.39%, and the amount of unsaturated fatty acids was 65.06, 85.78 and 86.61% of total fatty acids, respectively. The fatty acid profiles of the oil for the culture possibility met the necessary requirements and are, therefore, promising resource for biofuel production.