Environmental factors directly affect the growth and composition of microalgal biomass. Therefore, the present work analyzed the metabolomics (amino acids, organic acids, and fatty acids) of the microalga Scenedesmus obliquus cultivated in 24:0 and 12:12 (light:dark) photoperiods and different phases of cell growth. Furthermore, the metabolites were related to protein, lipid, and chlorophyll contents at the end of cultivation. The highest biomass concentration (4020 mg L- 1) and protein (47.3%) were obtained in culture under constant illumination. The cultivation 12:12 (light:dark) photoperiod triggered higher production of lipids (23.0%) and chlorophylls (26.4 mg g- 1) by S. obliquus. Microalgal metabolites were greatly affected by photoperiod and by phase of cell growth. Thus, metabolite production could be related to both the environmental conditions under which cultivation occurred and to the different concentrations of products (proteins, lipids, and chlorophylls) present in the S. obliquus biomass.