The production of biomolecules by microalgae has a wide range of applications in the development of various materials and products, such as biodiesel, food supplements, and cosmetics. Microalgae biomass can be produced using waste and in a smaller space than other types of crops (e.g., soja, corn), which shows microalgae’s great potential as a source of biomass. Among the produced biomolecules of greatest interest are carbohydrates, proteins, lipids, and fatty acids. In this study, the production of these biomolecules was determined in two strains of microalgae (Chlamydomonas reinhardtii and Chlorella vulgaris) when exposed to different concentrations of nitrogen, phosphorus, and sulfur. Results show a significant microalgal growth (3.69 g L−1) and carbohydrates (163 mg g−1) increase in C. reinhardtii under low nitrogen concentration. Also, higher lipids content was produced under low sulfur concentration (246 mg g−1). It was observed that sulfur variation could affect in a negative way proteins production in C. reinhardtii culture. In the case of C. vulgaris, a higher biomass production was obtained in the standard culture medium (1.37 g L−1), and under a low-phosphorus condition, C. vulgaris produced a higher lipids concentration (248 mg g−1). It was observed that a low concentration of nitrogen had a better effect on the accumulation of fatty acid methyl esters (FAMEs) (C16-C18) in both microalgae. These results lead us to visualize the effects that the variation in macronutrients can have on the growth of microalgae and their possible utility for the production of microalgae-based subproducts.