The self-assembly of a bichromophoric naphthalenediimide (NDI) into nanofibers showed efficient energy transfer (light-harvesting) to perylenediimide (PDI) molecules through a host-guest interaction, which can be visualized by microscopy using samples deposited on glass surfaces. In combination with atomic force microscopy, spectral, and polarization analyses, fluorescence imaging unveiled the inhomogeneity, packing defects, and relative spatial arrangement of PDI and NDI molecular units, which were found to affect the exciton mobility along the NDI nanofibers and the energy transfer efficiency from NDI to PDI guests. Fluorescence microspectroscopy shows that efficient energy transfer occurs from NDI nanofibers to isolated PDI molecules and their partial self-aggregates. The NDI nanofibers emit strong blue polarized emission, while the emission corresponding to PDI guest molecules is weakly polarized, indicating the local disruption of NDI chromophore-ordering upon PDI guest binding.