Standard and mutant Tisochrysis lutea strains were grown in batch culture for 10 days under nitrogen (N)-replete and N-reduced conditions to determine the effects of N supply in culture medium and growth phase on microalgal physiology and biochemistry. These two T. lutea strains were compared in terms of growth, morphology, associated free-living bacterial community, viability, intracellular lipid content (as measured by Bodipy staining), chlorophyll autofluorescence, and biochemical composition, with a focus on lipid class and fatty acid compositions. The standard strain (T) reached higher cell counts regardless of N supply in culture medium. In both T. lutea strains, microalgal final cell density was significantly lower in N-reduced medium. Carbohydrates were enhanced at stationary phase in both strains, regardless of N supply. The oleaginous strain (T +) accumulated triacylglycerols; whereas, the standard strain T accumulated alkenones as reserve lipids, especially in N-reduced medium and stationary phase. Each T. lutea strain exhibited an intrinsic specific FA profile in neutral lipids (NL) and, to a lesser extent, in polar lipids (PL) independent of N supply and growth phase. T + contained more saturated and monounsaturated fatty acids (especially 14:0 and 18:1n-9), but less polyunsaturated fatty acids (18:4n-3 and 22:6n-3) than T. Overall, growth phase induced more changes in fatty acid profiles of both T. lutea strains in NL and PL than N supply in culture medium.