Mixed sugar co-fermentation represents an interesting fermentation strategy to improve bioethanol production from low-cost raw materials, such as sugarcane bagasse. The aim of this study was to evaluate a mixture of sugars (hexoses and pentoses) from sugarcane bagasse hemicellulosic hydrolysate diluted with sugarcane molasses as substrate to be converted either in monoculture or co-culture fermentations. A co-culture fermentation technique using hexoses-fermenting yeasts (Saccharomyces cerevisiae: from Santa Adélia Mill, an industrial strain (SA) and a respiratory-deficient mutant strain (ScP)) together with xylose-fermenting yeasts (Scheffersomyces stipitis (SS) and Spathaspora passalidarum (SP)) was carried out to enhance bioethanol production. Co-culture fermentation in a medium with a high mixed sugar concentration (100 g/L) resulted in a complete sugar consumption, ethanol titer of 30.2 g/L and increased ethanol productivity (Qp =4.44 g/Lh) using wild type strains (SP+SA). In contrast, the maximum ethanol titer (49.2 g/L) was reached during co-culture performed with S. stipitis and the respiratory-deficient mutant strain of S. cerevisiae, however ethanol productivity (Qp =0.60 g/Lh) decreased due to the poor fermentation performance of the mutant strain. This work suggests that improved efficiency and good scalability of the mixed sugar fermentation process makes an important basis for the economic viability of bioethanol production from lignocelluloses.