Abstract Single-stage cultivation strategy is more favorable than two-stage cultivation owing to the lesser technical obstacles and being more cost-effective in the microalgal biodiesel production. Hence, in this study, mixotrophically grown Chlorella minutissima MCC 27 biomass was optimized for synergistic maximization of biomass yield and intracellular lipid accumulation using Central Composite Rotatable Design (CCRD). The effect of mix-carbon sources (glucose + acetate), nitrate concentration and culture time on biomass accumulation and lipid content (% DCW) was evaluated separately using Response Surface Methodology (RSM). Out of the three strategies, viz., biomass optimization (BO), lipid content (% DCW), and simultaneous production of biomass and lipid content and their maximization, the third strategy was found to be most effective to obtain the maximum lipid productivity (108.81 mg L−1 d−1), which was approximately 10-fold higher than the autotrophically grown C. minutissima culture. The optimized conditions for maximum lipid productivity were found to be 5.96 g L−1 glucose, 4.12 g L−1 acetate, 0.73 g L−1 nitrate and incubation period of 10 days. Palmitic acid was the principal FAME composite followed by oleic acid and the ratio of saturated fatty acids (SFA):monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) was found to be 1.9:1.8:1. Accordingly, simultaneous biomass and lipid content optimization was found to be a better strategy for the maximization of lipid yield as compared to the other strategies followed in the present study.