Microalgae are an attractive source of biomass for fossil fuel alternatives and renewable energy sources. Regardless of their potential, the development of microalgal biofuels has been limited due to the associated economic and environmental costs. We followed and compared the biomass properties of six Chlorellaceae strains with a specific interest in lipid-based biofuels. The strains were cultivated under balanced nutrient limitation inducing a gradual limitation of nutrients that triggered reserve accumulation. The final biomass of each strain was characterized by its elemental and biochemical composition. Due to its high lipid content and overall composition, Chlorella vulgaris NIES 227 was identified as an ideal feedstock for biofuels with the best energy-content biomass. Its fatty acid profile also showed superior qualities for biodiesel production. Balanced nutrient limitation promoted not only the accumulation of storage compounds in all strains, but also resulted in a low content of heteroatom precursors and ashes for biofuel applications.