The present work describes an experimental study about the shear strength and the mode I fracture toughness of adhesive joints with substrates pre-treated by pulsed laser ablation. An ytterbium-doped pulsed fiber laser was employed to perform laser irradiation on AA6082-T4 alloy. Morphological and chemical modifications were evaluated by means of surface profilometry, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Thick adherend shear tests were carried out in order to assess the shear strength while the mode I fracture toughness was determined using the double cantilever beam. For comparison, control samples were prepared using classical surface degreasing. The results indicated that laser ablation has a favorable effect on the mechanical behavior of epoxy bonded joints; however, while a+20% increase was recorded for shear strength, a remarkable threefold enhancement of fracture toughness was observed with respect to control samples. XPS analyses of treated substrates and SEM observations of the fracture surfaces indicated that laser pre-treatment promoted chemical and morphological modifications able to sustain energy dissipation through mechanical interlocking. As a result cohesive failure within the adhesive bond-line was enabled under predominant peel loading.