The utilization of reclaimed asphalt pavement (RAP) in flexible pavement construction and rehabilitation has gained significant traction, driven by the need to conserve limited natural resources. This study explores the effectiveness of bio-based oils derived from harvested crops as rejuvenators in asphalt mixtures containing 50% RAP. Comprehensive assessments were conducted, including penetration, softening point, viscosity and ductility tests, to determine the optimal rejuvenator dosage. Additionally, evaluations of rolling thin film oven (RTFO) aging, retained penetration and ductility, equivalent softening and breaking points, plasticity temperature range, stiffness modulus, complex modulus, and phase angle were performed. Fourier-transform infrared (FTIR) spectroscopy, Marshall immersion, and moisture susceptibility tests further validated the rejuvenator's efficacy in improving asphalt properties. The incorporation of 1.8% bio-oil significantly reduced the viscosity of the mixtures, enhancing workability. The rejuvenator effectively compensated for the loss of light components in aged binders, restoring the maximum and minimum temperature performance to levels comparable to virgin asphalt (VA). Mechanical testing revealed that bio-oil mitigated the aging effects of RAP asphalt, and the rejuvenated mixtures showed considerable improvements over both VA and non-rejuvenated RAP mixtures, where the rejuvenated specimens achieved 90.8 and 89.4% of retained strength index (RSI) values for 24- and 48-h immersion periods, respectively comparing with 88.9 and 86% post-immersion of VA for similar durations. These findings underscore the potential of bio-oil as a sustainable and eco-friendly solution for rejuvenating aged asphalts, paving the way for more sustainable asphalt production practices.