There is an increasing need for the development of alternative energy sources with a focus on reducing greenhouse gas emissions and striving toward a sustainable economy. Bioethanol and biodiesel are currently the primary choices of alternative transportation fuels. At present, biodiesel is not competitive with conventional fuel due to its high price, and the only way to compete with conventional fuel is to improve the quality, reduce the costs, and coproduce value-added products. With the high demand for lipids in the energy sector and other industrial applications, microbial lipids accumulated from microorganisms, especially oleaginous fungi and yeasts have been the important topic of many recent research studies. This chapter summarizes the current status of knowledge and technology about lipid production by oleaginous fungi and yeasts for biofuel applications and other value-added products. The chapter focuses on several aspects such as the most promising oleaginous strains, strain development, improvement of lipid production, methods and protocols to cultivate oleaginous fungi, substrate utilization, fermentation process design, and downstream processing. The feasibility and challenges during the large-scale commercial production of microbial lipids as fuel sources are also discussed. It provides an overview of microbial lipid production biorefinery and also future development directions.