Using atomic force microscopy for investigating the properties of asphalt has been common in recent years. In this study, the microstrucure of an asphalt cement modified by 3% (by weight) Sasobit and 3% ethylene vinyl acetate (EVA) has been studied using AFM. The binders have been studied in unaged condition and after being aged using rolling thin film oven test (RTFOT) and pressure aging vessel (PAV) to find the effect of aging condition and additives on the microstructure through the change in bee structures and surface roughness. Results revealed that the additives and aging changed the asphalt microstructure. In unaged condition, the density and number of bee structures, and the mean size and area of bee structures in modified binders are more than those of the base binder. The total area occupied by the bee structures for the Sasobit modified asphalt is less than that for the base binder; and for the EVA and Sasobit modified binder is higher than that for the base binder. While, the aging increases the total area, number and mean size of bee structures in the unmodified asphalt binder, it causes reduction of the number, density and total area of bee structures in modified binders. The roughness of samples’ surface reveals that the additives increase the surface roughness of the base binder, indicating that the stiffness is improved using the additives. Moreover, the roughness increases with aging in the base binder; however, in modified binders, the aging results in reduction of roughness indices; indicating that the modified binders are less sensitive to aging compared with the base binder.