© 2020 Elsevier Ltd Due to expensive culture realizations and low lipid yields, microalgae-based biodiesel production still lacks economic viability at an industrial-scale. Optimization of lipids and fatty acid methyl esters (FAMEs) distribution is a necessary condition to get sustainable microalgae biodiesel production. Nutritional sources (nitrogen, phosphorous, carbon and iron) influence the lipid accumulation and biomass production yield. The aim of this study is to get high-quality biodiesel by a microalgae-yeast co-culture based on the selection of culture media micronutrients’ distribution. A response surface-based combination of NaNO3, K2HPO4 and FeSO4*7H2O nutrients increases biomass and lipids accumulations. The obtained highest biomass and lipids contents are 1.68 gL−1 and 27.77%, respectively. Under the selected optimized micronutrients’ composition, high-saturated fatty acids are obtained if high ratio of N/P (6:1 and 11:1) and unsaturated fatty acid under N/P ratio of (9:1) are considered. These results suggest microalgae-yeast co-culturing under controlled N/P ratio in the culture medium produce desirable lipids distributions, yielding better biodiesel quality and equilibrium between biomass and lipids accumulation. The selected micronutrient combination (with the selected N/P ratio) leads to augment the lipids content which is transformed into biodiesel. Biodiesel quality (determined by cetane number, iodine value, cloud point, oxidation stability, viscosity), is evaluated considering microalgae lipids and FAMEs produced by transesterification process. Fourier transform infrared (FTIR) corroborates the lipids distribution, which can be used to produce high quality biodiesel. Gas chromatography (GC) verifies the production of desirable methyl esters (C16 and C18).