Lignocellulosic biomass is an abundant renewable source of xylose and glucose, which can be converted into ethanol using microorganisms. The aim of this study was to use sugarcane bagasse hydrolyzate (HBC) supplemented with sugarcane molasses "B", one of the liquors resulting from the crystallization step in cane sugar production process, in a mixed yeast culture (Saccharomyces cerevisiae ITV-01 and Scheffersomyces (formerly Pichia) stipitis NRRL Y-7124) in order to increase ethanol production and the complete utilization of hydrolyzate sugars. The culture of mixed yeast in a medium with 50% HBC supplemented with 8% v/v intermediate molasses "B" after 42 h fermentation exhibited a significant increase in ethanol production (53.80 gL(-1)), yield (0.45 gg(-1)) and productivity (1.07 gL(-1) h(-1)). The novel strategy of using 8% v/v sugar cane molasses "B", promotes complete sugar consumption and a 74% increase in ethanol fermentation by the mixed culture of S. cerevisiae ITV-01 and Scheffersomyces stipitis NRRL Y-7124. (C) 2014 Elsevier Ltd. All rights reserved.