A careful analysis of the magneto-transport properties of epitaxial nanostructured Nb thin films in the normal and the mixed state is performed. The nanopatterns were prepared by focused ion beam (FIB) milling. They provide a washboard-like pinning potential landscape for vortices in the mixed state and simultaneously cause a resistivity anisotropy in the normal state. Two matching magnetic fields for the vortex lattice with the underlying nanostructures have been observed. By applying these fields, the most likely pinning sites along which the flux lines move through the samples have been selected. By this, either the background isotropic pinning of the pristine film or the enhanced isotropic pinning originating from the nanoprocessing have been probed. Via an Arrhenius analysis of the resistivity data the pinning activation energies for three vortex lattice parameters have been quantified. The changes in the electrical transport and the pinning properties have been correlated with the results of the microstructural and topographical characterization of the FIB-patterned samples. Accordingly, along with the surface processing, FIB milling has been found to alter the material composition and the degree of disorder in as-grown films. The obtained results provide further insight into the pinning mechanisms at work in FIB-nanopatterned superconductors, e.g. for fluxonic applications.