The objective of this work was to evaluate the effect of different photo-cycles on the growth and biochemical profile of Scenedesmus obliquus CPCC05, focusing on food interest compounds. The photo-cycle conditions were separated into three groups: long-term photo-cycles (24:0, 22:2, 20:4, 18:6, 12:12, and 10:14 (h:h)), frequency photo-cycles (2, 4, 8, 12, 24, and 48 times per day (t/d)), and short photo-cycles (0.91:0.09, 0.83:0.17, 0.75:0.25, and 0.50:0.50 (s:s)) of light:dark, respectively. The results showed these microalgae can store enough energy to support cell growth for continuous periods of up to 2 h in the dark, without affecting the productivity of the process. This 2 h, when divided into 2 cycles per day (2 t/d), showed the best growth condition (3700 mg L−1), generation time (14.40 h), and maximum biomass productivity (21.43 mg L h−1). This photo-cycle of 2 t/d was also the best condition for the production of total sterols. However, the values of polyunsaturated fatty acids, lipid content, and amino acids obtained higher yields in the short photo-cycle of 0.75:0.25. Thus, the modulation of light cycles becomes an important tool for boosting and directing the production of target molecules in phototrophic cultures of microalgae.