We investigated the fermentation of a mixture of oat and soybean hulls (1:1) subjected to acid (AH) or enzymatic (EH) hydrolyses, with both showing high osmotic pressures (> 1200 Osm kg(-1)) for the production of ethanol. Yeasts of genera Spathaspora, Scheffersotnyces, Sugiytnaella, and Candida, most of them biodiverse Brazilian isolates and previously untested in bioprocesses, were cultivated in these hydrolysates. Spathaspora passalidartan UFMG-CM-469 showed the best ethanol production kinetics in suspended cells cultures in acid hydrolysate, under microaerobic and anaerobic conditions. This strain was immobilized in LentiKats (R) (polyvinyl alcohol) and cultured in AH and EH. Supplementation of hydrolysates with crude yeast extract and peptone was also performed. The highest ethanol production was obtained using hydrolysates supplemented with crude yeast extract (AH-CYE and EH-CYE) showing yields of 0.40 and 0.44 g g(-1), and productivities of 0.39 and 0.29 g (L h)(-1), respectively. The reuse of the immobilized cells was tested in sequential fermentations of AH-CYE, EH-CYE, and a mixture of acid and enzymatic hydrolysates (AEHCYE) operated under batch fluidized bed, with ethanol yields ranging from 0.31 to 0.40 g g(-1) and productivities from 0.14 to 0.23 g (L h)(-1). These results warrant further research using Spathaspora yeasts for second-generation ethanol production.