The aim of this study was to evaluate a temperature-induced two-stage cultivation (TTC) strategy for the regulation of lipid and carbohydrate production by two microalgae, Chlorella vulgaris and Dunaliella salina, for biofuel production. The microalgae were grown under several temperature conditions (15, 25, 35, and 45°C) and optimal growth was observed at 25°C for both microalgae. To test the TTC, aseptically cultured microalgae were incubated under optimal conditions (25°C) for 20 days, and then divided into four aliquots that were incubated at 15, 25, 35, and 45°C for 5 days. Similar but somewhat decreased growth rates were observed at the non-optimal temperatures (15, 35, and 45°C). In addition, while total lipid accumulation increased in a temperature-dependent manner in both microalgae, total carbohydrate increased with temperature in C. vulgaris but decreased in D. salina. However, for lipid and carbohydrate production, while the highest lipid productions of C. vulgaris and D. salina were observed at 25°C and 35°C, respectively, the highest total carbohydrate productions of C. vulgaris and D. salina were obtained at 15°C and 25°C, respectively. These results suggest that the TTC strategy may be easily and efficiently applied to bioprocessing for biofuel production.