The use of phytohormones in microalgal biofuel production is gaining interest due to their substantial effect on biomass and lipid enrichment under standard and nitrogen-limited conditions. In the present study, the collaborative effect of combination of selected phytohormones viz. zeatin (Z), indole acetic acid (IAA), and gibberellic acid (GA) was studied to optimize biomass and lipid production in microalga Acutodesmus obliquus under nitrogen-limitation using response surface methodology (RSM). Significantly higher biomass and lipid productivity was obtained with the supplementation of combination of Z (0.5 mg L-1), IAA (1.0 mg L-(1)), and GA (5.0 mg L-1) under nitrogen-limitation with 49.07% and 77.20% increase respectively as compared to optimized nitrogen control (ON). Zeatin was found to be the most significantly influencing phytohormone followed by GA in RSM data analysis. Present study elucidated the synergistic effect of combination of exogenous phytohormones in enhancing biomass and lipid productivity under nitrogen-limitation for microalgal biofuel production.