Nanomaterials design, synthesis and characterization are ever-expanding approaches towards developing biodevices or neural interfaces to treat neurological diseases. The ability of nanomaterials features, to tune neuronal networks morphology or functionality is still under study. In this work, we unveil how, when interfacing mammalian brain cultured neurons, iron oxide nanowires (NWs) orientation affects neuronal and glial densities, and network activity. Iron oxide NWs were synthesized by electrodeposition, fixing the diameter to 100 nm and the length to 1 μm. Scanning electron microscopy, Raman and contact angle measurements were performed to characterize the NWs morphology, chemical composition and hydrophilicity. Hippocampal cultures were seeded on NWs devices and after 14 days the cell morphology was studied by immunocytochemistry and confocal microscopy. Live calcium imaging was performed to study neuronal activity. Using random (R-NWs) a higher neuronal and glial cell densities were obtained compared with the control and vertical (V-NWs), while using V-NWs more stellate glial cells were found. R-NWs produced a reduction in neuronal activity while V-NWs increased the neuronal network activity, possibly due to higher neuronal maturity and a lower number of GABAergic neurons, respectively. These results highlight the potential of NWs manipulations to design ad hoc regenerative interfaces.