The biofuels production from alternative and renewable raw materials is mandatory to achieve sustainable growth based on a bioeconomy. Eucalyptus bark is a waste generated during the chemical manufacturing of Eucalyptus pulp that can be used as an alternative source of biomass, suitable for the production of biofuels. In this work, Eucalyptus nitens bark (ENB) was fractionated by organosolv treatment for ethanol production. For that, a Doehlert experimental design was carried out to evaluate the dependent variables: temperature (170-200°C), time (30-90 min) and ethanol-water percentage (50-80 %) on delignification of Eucalyptus bark. Organosolv process was suitable for the fractionation of E. nitens bark. After treatment, 74-93 % of glucan was recovered and 25-52 % of delignification was achieved. Delignified ENB was subjected to simultaneous saccharification and fermentation process for bioethanol production. The results showed that the variables temperature and time of organosolv process had significant influence on ethanol production. The organosolv pretreatment improved the ethanol yield from 32 to 99%. This work shows a suitable process for the valorization of Eucalyptus bark into bioethanol.