The effect of sugarcane molasses on lipid content and fatty acids methyl ester (FAME) profile by Chromochloris zofingiensis is investigated in this work. For this purpose, the strain has been cultivated under mixotrophic conditions in a medium amended with specific concentrations of molasses (0.5 g/L, 1 g/L, and 2 g/L) able to sustain microalgae growth. Better biomass concentration, lipid content, and lipid productivity (1.6 g/L, 38% wt, and 286 mg/L/day) than the control (1.32 g/L, 16% wt, and 139 mg/L/day) were obtained with 2 g/L of molasses. The highest value of the total lipid content (42% wt) was reached with 1 g/L of molasses. FAME profile revealed a 97% composition in C16-C18 with no statistically meaningful differences among the three concentrations of molasses tested. The most represented fatty acids were C18:1 oleic (> 38% wt), C18:2 linoleic (> 21% wt), and C16:0 palmitic (> 14% wt). When using 2 g/L of molasses unsaturated fatty acids reached the highest portion (77%) than the control (61%). Specifically, all FAMEs have been quantitatively and qualitatively analyzed in order to improve biodiesel properties. The main characteristics of biodiesel obtainable from the algal lipids appeared in compliance with ASTM standards for unblended biodiesel and fulfilled European regulations (EN 14,214 and EN 590) for the quality of biodiesel. Owing the compliance of the corresponding biodiesel characteristics with international standards, a profitable biodiesel can be obtained under mixotrophic conditions triggered by the use of a food industry waste. Graphical abstract: [Figure not available: see fulltext.]