Ethanol-water organosolv pulping has recently been reported as an effective method for biorefinery of woody substrates. The process yields carbohydrate-enriched substrates with very low content of unfermentable components and a high-quality lignin suitable for a wide range of industrial applications. In our study we proposed a modification of the classical organosolv pulping technology which included, as a first step, fungal biotretament of wood chips. Two wood species, a softwood, Pinus radiata, and a hardwood, Acacia dealbata, were biotreated with white-rot fungi (Ceriporiopsis subvermispora or Ganoderma australe) followed by an organosolv delignification with ethanol-water. Undecayed wood was used as control. Biotreated wood chips yielded pulps with higher glucan and lower lignin content compared to control samples. P. radiata and A. dealbata bio-pulps showed higher glucan (93% and 82%, respectively) and lower lignin content (6% and 13%, respectively) than control pulps (82% glucan, 13% lignin and 92% glucan, 4% lignin, respectively). Pretreated lignocellulosic substrates were readily hydrolyzed by a cellulase supplemented with a β-glucosidase. Ethanol fermentation of carbohydrate hydrolyzates was accomplished with a Saccharomyces cerevisiae yeast strain. The highest ethanol yield (>80%) was observed for hydrolyzates derived from bio-organosolv pulps. Therefore, preliminary biotreatment of wood combined with consecutive organosolv biomass fractionation demonstrates a number of advantages over the classical organosolv pulping. In particular, it was observed higher overall ethanol yields at milder substrate pretreatment conditions resulting in lower energy consumption, higher overall carbohydrate recovery, and lower generation of enzyme and yeast inhibitors.