Fermentation of carbohydrates to produce bioethanol is one of the pathways to produce biofuels from microalgae. This process currently needs many stages that are complex and energy consuming. Cell wall disruption and hydrolysis are two of the stages that must be carried out, since most carbohydrates are entrapped within the cell wall or intracellularly as energy storage in the form of starch. In the present work, physical, chemical, and enzymatic pre-treatments were performed on three microalgal species to disrupt and break down complex carbohydrates into simple sugars, as a preliminary stage to produce bioethanol. Pre-treatments were carried out alone and combined with each other. According to the results obtained in the present work, the highest concentration of monosaccharides per g of microalgae dry weight was achieved by the combination of pre-treatments; for Chlorella sorokiniana and Nannochloropsis gaditana the combination of acid hydrolysis followed by enzymatic hydrolysis produced 128 and 129 mg/g, respectively. In the case of Scenedesmus almeriensis the highest monosaccharide concentration (88 mg/g) was obtained after acid hydrolysis with sulphuric acid for 60 min at 121 degrees C. The results obtained proved the effectiveness of the combination of acid pre-treatment and enzymatic hydrolysis to enhance complex carbohydrates break down into simple-sugars in bioethanol production process from microalgal biomass. (C) 2014 Elsevier B.V. All rights reserved.