This study evaluated the use of a renewable organic acid (citric acid) as homogeneous catalyst for the pretreatment of raw sugarcane bagasse (SB). A 22 central composite design with two factors (reaction time and citric acid concentration) was used to evaluate the effect of the factors on the chemical composition of the pretreated SB samples and enzymatic hydrolysis efficiency. The enzymatic hydrolysis experiments of the pretreated SB samples were performed using the enzymatic cocktail Cellic® CTec 3 and the total reducing sugars (TRS) released after enzymatic hydrolysis was determined. In addition, scanning electron microscopy and X-ray diffraction were used to examine the changes in the morphology and structure of the pretreated SB samples. The changes in the structure and chemical composition of the pretreated SB resulted in a high TRS concentration (28.2 g L−1) after enzymatic hydrolysis, for the pretreatment condition employing 6 wt% citric acid and 102.4 min, compared to the raw SB (3.06 g L−1). The pretreatment showed potential for application since a moderate conversion of polysaccharides in TRS was obtained, even though it was a pretreatment carried out under milder conditions. The pretreatment studied has the advantage of using weak and environmentally friendly acid catalyst, atmospheric pressure, and low temperature.