With continuous economic and technological growth, sustainable development must be pursued and comprehensive transformation of economic and social development systems must promoted to allow humans and nature to coexist harmoniously. The use of renewable energy to replace traditional fossil materials in road engineering can reduce carbon emissions and serve as an environmentally friendly approach. Bioasphalt production and the use of biological rejuvenators in road areas have attracted research attention. Existing studies show that biological rejuvenators can effectively improve the regeneration efficiency of reclaimed asphalt pavement (RAP). Different biological rejuvenators have different mechanisms for RAP recovery, which determine the recovery efficiency of RAP. Based on an analysis and summarization of the application of different types of rejuvenators, we provide a comprehensive review of the current studies on biological rejuvenators applied to roads, including the mechanisms, performance, and challenges associated with rejuvenators from different biological sources. Additionally, we discuss the potential for using microorganisms as asphalt rejuvenators. This review shows that plant-sourced rejuvenators can supplement the missing light components in aging asphalt at the colloidal structural level via diffusion and fusion to restore the original asphalt properties. Animal- and algal-sourced rejuvenators can depolymerize the associations formed by van der Waals forces, hydrogen bonds, charge transfer, and π-π interactions among aromatic lamellae in aged asphalt through intermolecular interactions. Microorganisms can crack polycyclic aromatic hydrocarbons (PAHs) at the molecular level and have potential as asphalt rejuvenators. This review provides a reference for the future application of biological rejuvenators from different sources to road engineering.