In this work, the analytical potential of cyclical electrical field flow fractionation (CyElFFF) for nanomaterial and colloidal particle characterization has been experimentally demonstrated. Different operating parameters were investigated in order to evaluate their effect on the mechanisms of retention and fractionation power of CyElFFF. The voltage and frequency of the oscillating electrical field appeared to be the most influential parameters controlling the separation mode. Mobile phase flow rate was also found to be a key parameter controlling the fractionation efficiency. This work allowed the definition of operating conditions such that a reliable CyElFFF analysis could be performed on different nanoparticles on the basis of the direct comparison of their theoretical and experimental behavior. The results show that this technique in optimized conditions is a powerful tool for electrophoretic mobility based separation and characterization of various nanoparticles.