Charge generation, recombination, and transport in organic semiconductors are fundamental processes that dictate the performance for a range of organic devices. Here we used electrostatic force microscopy combined with laser scanning microscopy to study the carrier distribution in individual organic nanofibers that result from localized illumination: NaT2 (5,5′-bis(naphth-2-yl)-2,2′-bithiophene) crystalline nanofibers were illuminated with a continuous wave, focused laser beam with above-band gap photon energy. The localized illumination causes a local negative charge accumulation at the illumination position, while the remaining part of the nanofiber exhibits a positive charge. We propose that this inhomogeneous distribution of photogenerated holes and electrons along the nanofiber axis is due to a surface-trap induced exciton dissociation and the difference in charge carrier mobilities for hole and electrons. We use a one-dimensional, drift-diffusion based model to rationalize the observations and give an improved understanding of the factors governing the charge build-up.