Neuronal cell cultures have been instrumental to the advance of our knowledge in a wide range of areas. One of the limitations of this model is the lack of stability, over time, of the substrate materials that are used in neuronal cell cultures. In this work, sputtering was used to permanently modify glass coverslips with a polyamide 6.6 thin film, using different discharge gases. Differences in the chemical composition of the films, resulting from the conditions used in deposition, were correlated with the surface properties. After characterization by SEM, TEM, AFM, XPS, zeta-potential, wettability and evaluation of the mechanical properties, some surfaces were selected for in vitro testing in the development of cultured cerebrocortical neurons. The results showed that the chemical composition and surface charge are particularly affected by the variation of the deposition parameters, although wettability, for example, did not present significant variations. In vitro tests revealed that the surfaces with higher NH2/COOH ratio allowed the adhesion and differentiation of neurons, with a morphology similar to that obtained after plating of the cells on poly-D-lysine-coated glass. This work opens new perspectives for the permanent modification of materials towards multifunctional neural compatible surfaces and open new areas for bioengineering applications.