Eucalyptus biomass was studied as a feedstock for sugars release using an alkaline extrusion plus a neutralization-based pretreatment. This approach would be a first step in a bioconversion process aimed at obtaining fuel bioethanol from eucalyptus biomass. The best operation conditions of extrusion (screw speed, temperature, liquid to solid ratio and NaOH amount) that lead to an effective destructuration of lignocellulose and enhanced sugar release were investigated. Two process configurations, with and without filtration inside the extruder, were tested. In the case without filtration, washed and not washed extrudates were compared. It was demonstrated that filtration step was convenient to remove inorganic salts resulting from neutralization and to promote the mechanical effect of extrusion, but limitations in the machine used in the work prevented testing of temperatures above 100 °C using this configuration. In the no filtration strategy, a temperature of 150 °C allowed attaining the highest glucan and xylan conversion rates by enzymatic hydrolysis of extruded biomass, almost 40% and 75%, respectively, of the maximum yield that could be attained if all carbohydrates contained in raw eucalyptus were converted to sugars. Some of the mechanisms and individual effects underlying alkaline extrusion of eucalyptus were figured out in this work, providing guidelines for a successful pretreatment design that needs to be further studied.