Aging of asphalt is a major cause of pavement distress. While regenerators restore aged asphalt, their mechanisms and efficacy differences remain unclear. This study quantified the repair effects of waste bio-oil (WBO) and mineral oil (MO) rejuvenators on aged asphalt binder using a comprehensive characterization approach. Conventional properties (penetration, softening point, ductility), functional groups (FT-IR), thermal stability (TG), differential scanning calorimetry (DSC), and dynamic shear rheology (DSR) were analyzed. Results reveal distinct mechanisms: WBO acts chemically via polar molecules, selectively reducing oxygen-containing groups and significantly improving ductility, while MO acts physically through light components that dilute viscosity, exhibiting weaker chemical repair. WBO-regenerated asphalt showed a lower thermal-oxidative peak temperature, superior low-temperature ductility, and enhanced high-temperature rheological performance (higher rutting factor, optimized viscoelasticity). These mechanistic differences—chemical restoration (WBO) versus physical replenishment (MO)—determine performance outcomes at the binder level. The findings provide a theoretical basis for regenerator selection in pavement engineering, highlighting WBO’s advantages for functional group restoration and balanced thermal rheological properties, supporting sustainable road development.