Contact interaction of neuronal cells with extracellular nanometric features can be exploited to investigate and modulate cellular responses. By exploiting nanogratings (NGs) with linewidth from 500 nm down to 100 nm, we here study neurite contact guidance along ultra-small directional topographies. The impact of NG lateral dimension on the neuronal morphotype, neurite alignment, focal adhesion (FA) development and YAP activation is investigated in nerve growth factor (NGF)-differentiating PC12 cells and in primary hippocampal neurons, by confocal and live-cell total internal reflection fluorescence (TIRF) microscopy, and at molecular level. We demonstrate that loss of neurite guidance occurs in NGs with periodicity below 400 nm and correlates with a loss of FA lateral constriction and spatial organization. We found that YAP intracellular localization is modulated by the presence of NGs, but it is not sensitive to their periodicity. Nocodazole, a drug that can increase cell contractility, is finally tested for rescuing neurite alignment showing mild ameliorative effects. Our results provide new indications for a rational design of biocompatible scaffolds for enhancing nerve-regeneration processes.