The dc electrical characterization of single electrospun ZnO nanofibers (NFs) calcined at various temperatures was investigated. The linearity of Ti/Au ohmic contacts was confirmed for a wide range of voltage values. Parameters such as dc fibers resistivity in an atmosphere of dry air and nitrogen were determined. The studies have shown that the entire volume of fiber is depleted of charge carriers. The I–V dependence of fibers and contacts showed a linear behavior for a wide range of temperatures. Temperature dependence of conductivity was evaluated. Arrhenius plots revealed that the electrical conduction is mainly thermally assisted in the extended states. The activation energy was found to be strongly dependent on the grain size, which in turn depends on the fiber annealing temperature. This could be caused by segregation of point defect in nanocrystalline ZnO and changes in carrier concentration. To explain this effect, the authors proposed a model of donor depletion in grain based on the Mott–Schottky approximation. To the best of our knowledge, we report the first systematic study related to electrical characterization of single electrospun ZnO NFs, calcined at various temperatures, which allows for estimation of resistivity and activation energies in dry air and nitrogen atmosphere.