The strategic microalgal lipid production without growth reduction is the desideratum for sustainable microalgal biodiesel production. The present study investigated the effect of polysorbates (polysorbate 20, polysorbate 40, polysorbate 60 & polysorbate 80) supplementation on growth, biomass and lipid production of microalga Tetradesmus obliquus BPL16. T. obliquus BPL16 at controlled condition showed 12.5% dry cell weight (DCW) of lipid content and 0.8 g L⁻¹ of biomass production. Each of the four tested polysorbates at various concentrations magnificently influenced the green microalga with a maximum lipid production of 47.1% DCW and 46.5% DCW achieved at 0.15% of polysorbate 80 and 0.1% of polysorbate 60 supplementations respectively. The maximum biomass (2.6 g L⁻¹) production was achieved at 0.15% of polysorbate 40 and polysorbate 60 supplementation respectively. In addition, neutral lipids and vital fatty acids proportion increased at high lipid productive conditions enabled by polysorbate supplementation.