In recent years, the use of bio-based additives and recycled materials in asphalt pavements has attracted considerable attention due to their potential to improve mechanical performance while reducing environmental impacts. This study investigated the individual and combined effects of lignin and cellulose nanofiber on the mechanical properties and microstructural characteristics of hot mix asphalt. In addition, the behavior of mixtures containing 50%reclaimed asphalt pavement was comparatively evaluated. For this purpose, lignin contents of 10%, 15%, and20% by weight of binder and CNF contents of 0.5%, 0.75%,and 1.0% by weight of binder were incorporated into the mixtures. The mechanical performance of the mixtures was evaluated using indirect tensile strength, dynamic creep, four-point bending beam fatigue, and low-temperature fracture resistance tests. The results indicated that lignin increased mixture stiffness and enhanced resistance to permanent deformation, whereas CNF exhibited a more pronounced effect on improving fatigue performance and low-temperature fracture resistance. The simultaneous use of lignin and CNF provided a more balanced combination of stiffness and flexibility in the asphalt mixtures. Fracture resistance results showed that increasing CNF content enhanced fracture energy and reduced mixture brittleness, while higher lignin contents reduced fracture toughness. Comparative evaluation of RAP-containing mixtures revealed that the presence of aged binder slightly improved rutting resistance but reduced fatigue and low-temperature cracking resistance. Microstructural analyses further demonstrated that increasing the dosage of either additive reduced binder homogeneity and increased the tendency for particle agglomeration. Based on the overall performance evaluation, the mixture containing approximately 10%lignin and 0.5–0.75%CNF exhibited the most favorable balance among the investigated performance indicators under different temperature conditions. The findings suggest that the controlled combined use of lignin and CNF can effectively balance rutting resistance, fatigue performance, and low-temperature cracking resistance, thereby providing a promising approach for the development of sustainable asphalt mixtures incorporating recycled materials.