Nowadays, the growing energy costs and pressing worldwide demand for petroleum-based products create a strong need to develop alternative binders deriving from green and renewable sources. Bio-binders (or bitumen added to bio-based materials) can potentially be a viable alternative for the production of bituminous mixture, promoting the circular economy as well as environmental sustainability principles without reducing the overall performance of the mixture. In this context, the current study focuses on evaluation of the effects of a bio-binder on the mechanical response of asphalt concrete (AC) produced with it. In particular, a 10% bio-oil deriving from a by-product of the paper industry has been blended with a conventional 50/70 penetration grade bitumen to obtain the bio-binder. Moreover, plain bitumen having the same consistency was chosen to produce a reference AC. Two dense-graded AC wearing courses were prepared in the laboratory according to Italian technical specifications. A mechanical characterization in terms of indirect tensile strength, indirect tensile stiffness modulus, fatigue response and permanent deformation resistance was performed on gyratory compacted specimens using both conventional and performance tests. In addition, aging and water sensitivity of the AC specimens were evaluated. Overall results highlight that the AC produced with the bio-binder did not show reduced mechanical properties and it was comparable to the reference AC regardless of aging and water conditioning. This highly encourages the use of bio-binder as a viable alternative in asphalt technology.