Multi-scale evaluation of the rheological and mechanical properties of asphalt binder has substantial importance in understanding the binder’s micro- and macro-scale properties. This study compares the macro- and micro-scale mechanistic properties of asphalt binders. Test samples used in this study include performance grade binders (PG 64-22) from two different sources along with their modified counterparts. The modifiers include polyphosphoric acid (PPA), styrene-butadiene-styrene (SBS), a combination of SBS and PPA, and reclaimed asphalt pavement. To achieve the goal of this study, atomic force microscope technology was utilized to estimate the asphalt binder’s micro-mechanical properties (e.g., Derjaguin, Muller, Toropov modulus and deformation). On the other hand, data on the macro-scale properties—such as rutting factor (G*/sinδ), consistency and penetration—of the selected binders were analyzed and compared with the aforementioned micro-level properties. The comparative analyses indicated that the micro-mechanical properties of asphalt binders followed a linear trend with the macro-scale properties. The findings of this study are expected to help researchers and pavement professionals in modeling asphalt materials when multi-scale effects are deemed to be necessary.