Lignocellulosic biomass is an abundant industrial renewable source of xylose and glucose, which can be converted into bioethanol using alcoholic yeasts. Here, it is shown investigative procedures aiming to improve mixed sugar fermentation performance using sugarcane bagasse hydrolysate supplemented with molasses. In order to improve bioethanol production and complete utilization of hydrolysate sugars, single and co-culture fermentations with xylose (Scheffersomyces stipitis Y-7124 [SS]; Sapathapora passalidarium Y-27907 [SP]) and hexoses-fermenting yeasts (Saccharomyces cerevisiae Santa Adélia [SA]) were evaluated, at different mixed sugar concentration (50-100 g.L-1). The results indicated that the use of co-cultures fermentations with SP and SA ensured faster processes than single cultures of SS or SP. However, the rapid production of bioethanol by SA inhibited xylose-fermenting yeast, reducing fermentation performance. Moreover, SA re-assimilate bioethanol produced after all hexose content be depleted, probably due the presence of low levels of oxygen (necessary to allow xylose consumption by SS and SP). Despite of this problems, co-cultures with SP and SA in a medium with 50% of hydrolysate supplemented with 50% of molasses (100 g.L-1) promote complete sugar consumption after 50 h, exhibiting a significant increase in ethanol productivity (4.44 g.L-1.h-1), even with the presence of 2.5 g.L-1 of acetic acid.