Hydrolysis of cellulose by Trichoderma cellulases often results in a mixture of glucose, cellobiose, and low-mol-wt cellodextrins. Cellobiose is nonfermentable for most yeasts, and therefore it has to be hydrolyzed to glucose by beta-glucosidase prior to ethanol fermentation. In the present study, the beta-glucosidase production of one Penicillium and three Aspergillus strains, which were previously selected out of 24 strains, was investigated on steam pretreated willow. Both steam-pretreated willow and hemicellulose hydrolysate, released during steam explosion of willow, were used as carbon sources. Reference cultivation runs were performed using prehydrolyzed Solka Flee and glucose. The four strains were compared with Trichoderma reesei regarding sugar consumption and beta-glucosidase production. Aspergillus niger and Aspergillus phoenicis proved to be the best enzyme producers on hemicellulose hydrolysate. The maximum beta-glucosidase activity, 4.60 IU/mL, was obtained when A. phoenicis was cultivated on the mixture of hemicellulose hydrolysate and steam-pretreated willow. The maximum yield of enzyme activity, 502 IU/g total carbohydrate, was obtained when Aspergillus foetidus was cultivated on the hemicellulose hydrolysate.