In this work we showed the promising perspectives offered by the radiofrequency plasma processes on polymeric substrates. Polystyrene (PS) films with micropatterned grooves and nanostructured roughness were developed by Oxygen plasma treatment coupled with mask, and t process parameters, as power supply and treatment time were modulated. Then, hydrogenated amorphous carbon (a-C:H) coatings (∼30 nm thickness) were deposited by methane radiofrequency plasma enhanced chemical vapor deposition (rf-PECVD) on the polymer surface. Oxygen modified PS surface showed improved wettability, roughness and etching rate by increasing the power supply and the treatment time. Uniform and patterned bi-layer films show a regular surface morphology, uniform chemical properties, with a contact angle to water of 77°, a surface energy of 51.15 mN m−1 and good stability in physiological conditions. Nanoindentation measurements revealed a decrease of the bi-layer friction coefficient from 0.76 of PS to 0.17, highlighting the improvement of the nanomechanical properties of the novel developed system. Interaction with human bone-marrow mesenchymal stem cells demonstrates that uniform and patterned PS based films are biocompatible surfaces and remarkable, that groove patterned substrates induce stem cell alignment and elongation. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40427.