Yarrowia lipolytica is one of the most extensively studied ‘‘non-conventional’’ yeasts, being a strictly aerobic microorganism capable of producing important metabolites and having an intense secretory activity, which justifies efforts to use it in industry (as a biocatalyst), in molecular biology and in genetics studies. Moreover, Y. lipolytica has been considered an adequate model for dimorphism studies in yeasts. Yarrowia lipolytica presents the ability to grow on Olive Mill Wastewater (OMW) as well as to degradate organic compounds, including aliphatic and aromatic hydrocarbons, often accompanied by biosurfactants production. One of the most important products secreted by this microorganism is lipase which can be exploited for several applications in the detergent, food, pharmaceutical, and environmental industries. In addition, Y. lipolytica is able to produce citric acid and aroma from a variety of carbon sources, including sugars, alkanes, plant oils, starch hydrolysates, ethanol, and glycerol. Thus, this chapter presents an overview of Yarrowia lipolytica features and its major biotechnological applications.