Microalgae are used as a third-generation biofuel feedstock to overcome the problems, of first- and second-generation feedstocks, such as food versus fuel competition and longer harvesting time. However, the use of microalgae as a feedstock for biodiesel production is limited by the lipid content in the biomass. The development of strategies to enhance both the biomass and lipid content would lead to low-cost processes for biofuel production. In the present study, NaNO3, K2HPO4, NaCl, and initial medium pH were optimized to enhance both biomass yield and lipid content of freshwater microalgae Scenedesmus obliquus NCIM 5586 in a 5-L open pond system under light and dark period using central composite design. A multiobjective optimization method is employed to find the tradeoff between biomass and lipid accumulation and to predict the optimum composition for the multiple responses. The maximized yield of biomass and lipid was observed when concentrations of NaNO3, K2HPO4, and NaCl were 1.60 μM, 178.38 μM, and 24.80 mM, respectively, with initial medium pH of 7.23. The final yields of biomass and lipid at optimized conditions were recorded to be 1275.8 mg/L/day and 50.66% dcw, respectively. The obtained results indicate the significance of Scenedesmus obliquus cultivation in the open pond for the generation of feedstock for 3G biofuels.