Sugarcane bagasse, Agave tequilana bagasse and coffee husks were hydrolyzed by steam explosion followed by enzymatic treatment without detoxifying process prior to fermentation. The hydrolysates underwent sequential fermentations using three different microorganisms to produce bioethanol and xylitol. Sugarcane bagasse hydrolysate was found to be the best media for bioethanol (0.44 g/g yield, 85.5% efficiency) and xylitol (0.29 g/g yield) production and S. cerevisiae ITV01 performed efficient alcoholic fermentation in the presence of a high acetic acid concentration (5.0 g/L, initial hydrolysate concentration). For all hydrolysates, Candida tropicalis grew less; however, it produced more xylitol than Candida magnolia (0.29 and 0.22 g/g, respectively). The effect of oxygen on xylitol production in Candida tropicalis was also investigated, xylitol production improving as oxygen concentration level increased (flask volume/medium volume ratios of 1.25, 3.3 and 6.6 yielded 0.143, 0.320 and 0.406 g/g, respectively). The maximum calculated efficiency for the conversion of consumed xylose to xylitol was 44.6%, with a maximum xylitol production of 12.18 g/L.