Recently, the feasibility of using bio-materials to reduce or completely replace the use of bitumen in asphalt mixture has gained increasing attention. Amongst others, an interesting solution is represented by the use of wood co-products with mineral or vegetable oils. This research focuses on the development of bio-binders using vegetable resin (VR) in unmodified form and waste olive oil (WOO) as the main components; in order to optimize the rheological properties of the blends, crumb rubber from end-of-life tyres (CR), Styrene-Butadiene-Styrene (SBS) and polyethylene waxes (PEW) are used as additives. In particular, this investigation focuses on studying different oil/rosin ratios and polymer contents to provide a clear framework on this bio-binder solution; conventional bituminous binders are taken as a reference. The alternative binders are characterized in terms of conventional properties such as penetration depth and softening point, as well as rheological response. Finally, two of the bio-binders studied are selected with the aim of assessing the mechanical properties of the resulting sustainable asphalt mixture using the Marshall Stability test and the Indirect Tensile Strength test, comparing the results with the threshold values set by an Italian road agency. Thus, this research represents a preliminary analysis of the potential application of bio-binder mixtures within the specification limits imposed by road agencies. Although this research represents a first attempt, the results are promising and prove to be worthy of further investigations.