Increasing microalgae carbohydrate content through nutrient limitation has been regarded as a potential strategy to optimize microalgae bioethanol production. This study assessed three different media in which nitrogen, phosphorus and sulfur concentration was decreased by 10-folds with regard to the control media. The performance of two robust microalgae, namely Chlorella vulgaris and Scenedesmus sp., were evaluated in terms of growth and biochemical composition. At the stationary growth phase, sulfur and phosphorus limited media supported the highest carbohydrate accumulation on Chlorella vulgaris and Scenedesmus sp., respectively (approximately 4-fold). Under these scenarios, Scenedesmus biomass did not exhibit arrested growth while Chlorella decreased its concentration by 10%. This decrease was attributed to the decreased photosynthetic performance that concomitantly affected nutrients uptake. After 15 days cultivation, the biochemical analysis revealed that out of the total carbohydrates obtained under those limited media (30% DW for Chlorella and 22% DW for Scenedesmus), only 5% was starch and therefore the main fraction of carbohydrates was contained in the cell wall. Likewise, the carbohydrate profile presented fast changes in short periods of time and therefore the importance of an appropriate harvesting schedule to obtain carbohydrate rich biomass was highlighted.