Lignin is one of major by-product from wood and paper industry. As natural biopolymer, it could be a valid alternative as bitumen replacement and/or modifier to enhance the relevant performance of asphalt mixtures, promoting concurrently environmental benefits. In this context, the study focused on the effects of two different binders containing 30% of lignin (by weight) on the mechanical properties of bio-asphalt mixtures. Specifically, two different lignins were blended with two plain bitumens having different consistencies so obtaining two bio-binders with a similar consistency to that of a reference plain bitumen. The two bio-binders and the reference bitumen were used to produce in the laboratory three dense graded asphalt mixtures for binder courses according to the Italian Technical Specifications. The binder-aggregate adhesion, Indirect Tensile Strength, Stiffness Modulus, thermal susceptibility, fatigue resistance, and low-temperature cracking of mixtures were analysed in both unaged and long-term aged conditions. Overall, this investigation demonstrates that bio-mixtures containing lignin offer comparable results to the reference asphalt mixture. Moreover, the bio-asphalt mixtures exhibit a lower thermal susceptibility, as well as lower aging susceptibility. The findings of this research highly encourage the use of lignin as partial replacement of bitumen in asphalt pavements.