In this study, two indigenous Chlorella strains were evaluated for triacylglycerol (TAG) production and related parameters during 10 days of N-deprivation. The strains were identified based on morphological characteristics and genomic information, named as Chlorella sp. IMU12 and Chlorella sp. IMU17. Time-dependent reduction of growth was accompanied by decreased chlorophyll content, reduced oxygen evolution ratio, and elevated carotenoid content of both strains cultivated in N-free Bold's basal medium. Nitrogen deprivation induced total lipid and neutral lipid content in both strains, supported by FTIR measurement of TAGs. Chlorella sp. IMU17 showed higher production of TAGs as a response to N-deprivation. Strikingly, relative polysaccharide content showed a rapid increase on the first days and a noticeable reduction was recorded especially after 5 days of N-deprivation while TAG production showed a gradual increase during the whole period of N-deprivation in both strains. Starch might be a predominant form of carbon storage in the short term and the continuous increase of TAG production might be supported by degradation of starch in a longer period of N-deprivation. FAME analysis of lipids showed that the saturation level of the fatty acids of both strains meets the European standard EN 14214 requirements. Lastly, N-deprivation stimulated a significant reduction of PUFA production in Chlorella sp. IMU17. Thus, Chlorella sp. IMU17 might stand as a promising candidate for biodiesel feedstock production with its low PUFA content and a concomitant induction of saturation of fatty acids as a clear response to N-deprivation.