The increase of greenhouse gases into the atmosphere represents today one of the most global concern. The inevitable depletion of fossil fuels and the adverse climate changes push the scientific community to seek renewable and sustainable sources of fuel. In this scenario microalgae can be potentially exploited as renewable and environmentally friendly fuel resources. Wastewaters (WW) can be used as culture media reducing the costs associated to their cultivation. Hence, the goal of this study was to examine the effect of an organic rich WW on Chlorella vulgaris growth and fatty acid methyl esters (FAME) profile. This strain shows high biomass productivity thriving in a wide range of WWs and is able to shift its metabolism from autotrophic to hetero/mixotrophic one. Glycerol can be used to convey metabolism towards lipids production. Therefore, C. vulgaris was cultivated in dairy waste (DWW) with different concentrations of glycerol under both metabolisms. When C. vulgaris was cultivated under mixotrophy attained a high biomass yield compared to heterotrophy. The highest biomass yield (1.72 g L-1) was obtained with 10 mL of glycerol in DWW compared to the control (1.08 g L-1). When a two-phase metabolism was adopted, that is the first ten days under mixotrophy followed by the last five days in heterotrophy (MHD), the biomass was almost doubled with 2 mL of glycerol in DWW. FAME profile reveled that compared to the control the highest saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA) content were obtained under heterotrophy with 10 mL of glycerol, under MHD with 2 mL and with 4 mL of glycerol (46.73%wt, 41.79%wt, and 30.34%wt, respectively). A preliminary analysis on the saturated and unsaturated components of the FAME suggests that lipids extracted from C. vulgaris biomass cultivated mixotrophically and heterorophically in DWW could represents a feedstock to be exploited for biodiesel production.