In conclusion, this work demonstrates the efficient light-harvesting property of supramolecular nanofiber assemblies of (R)-1NDI towards (S)-2PDIagg guest molecules, as visualized by fluorescence microscopy. The lower relative fluorescence efficiency of energy transfer in the deposited samples compared to that in the bulk solution is attributed to inhomogeneities and packing defects present in the (R)-1NDI nanofibers, as evidenced by AFM and spectroscopy analyses. Uneven distribution of (S)-2PDI molecules on the (R)-1NDI nanofibers resulted in energy transfer from (R)-1NDI fibers to isolated PDI ((S)-2PDImono) as well as their partially self-aggregates ((S)-2PDIagg). Fluorescence polarization imaging revealed a strong dependence of polarized emission on nanofiber orientation. The nanofibers emit strongly polarized blue emission, indicating that the (R)-1NDI molecular units are stacked perpendicular to the long axis of the fibers. Weakly polarized emission at emission wavelength region higher than 500 nm indicates that the binding of (S)-2PDImono to the nanofibers has no preferred orientation, while its spherical self-aggregates (S)-2PDIagg interact more efficiently with the periphery of the fibers, which exhibit partial alignment, explaining the heterogeneity and differential energy transfer efficiency of our light-harvesting system. This finding highlights the importance of microscopy with spectral and polarization analysis for the comprehensive characterization of multicomponent light-harvesting systems and the identification of key features such as disorder, heterogeneity, and anisotropy in their interfaces, which can be manipulated/controlled to enhance the performance of future optoelectronic materials.