A second-generation polyphenylene dendrimer 1 self-assembles into nanofibers on various substrates such as HOPG, silicon, glass, and mica from different solvents. The investigation with noncontact atomic force microscopy (NCAFM) and scanning electron microscopy (SEM) shows that the morphology of the dendrimer nanofibers highly depends on substrate, solvent, and preparation method. Fluorescent nanofibers can be prepared from a polyphenylene dendrimer with chromophores either attached to the periphery of the dendrimer or incorporated in its core. Fluorescent nanofibers formed from polyphenylene dendrimer 4 with a perylenediimide core show isolated-chromophore emission due to the shielding of the rigid polyphenylene dendrons. Dendrimer 2 with one perylenemonoimide attached to its periphery self-assembles into fluorescent nanofibers, which exhibit a dimer like emission as a result of the interactions between peripheral chromophores. Dendrimer 3 with two perylenemonoimides at the rim does not form nanofibers by itself, but mixing of nonfluorescent dendrimer 1 with fluorescent dendrimers 2 or 3 leads to the formation of nanofibers with a homogeneous composition. Therefore, mixing can not only coassemble nonnanofiber-forming dendrimer 3 into nanofibers but also give rise to isolated-chromophore emission upon proper dilution.