Detailed structural, microstructural, biofilm formation and cytotoxicity studies were performed on Ti–Si–C–ON hard coatings prepared by DC reactive magnetron sputtering, in order to evaluate the relation among these properties. Compositional analysis showed the existence of two distinct regimens; regime I: high C/Si atomic ratio (C/Si≥1.42) and intermediate N/Ti atomic ratio; regime II: low C/Si atomic ratio (C/Si≤0.49) and low N/Ti atomic ratio. The structural analysis revealed that, in regime I, films crystallized in a B1-NaCl crystal structure typical of TiC0.2N0.8. In regime II, the decrease of C/Si and increase in silicon concentration led to the formation of Ti–Si–C–ON along with a reduction of grain size in the films. Atomic force microscopy observations showed that the surface morphology of these Ti–Si–C–ON films became smoother when the silicon content increased and the nitrogen content decreased, which is consistent with the formation of nanosized clusters. Concerning biological properties, it was observed that cytotoxicity could be related with the titanium concentration while biofilm formation ability was found to be related with the surface morphology of the films.