Functional nanocomposite coatings comprised of periodically stacked nanolayers of diamond-like carbon (DLC) and amorphous silicon were developed for biomedical applications. The periodical nanocomposite structure provided high surface durability while silicon aided in reducing the residual stress. The structural, mechanical, tribological, and biomedical properties of the Si/DLC coatings deposited by magnetron sputtering were investigated systematically. The effect of the negative substrate bias on the structure and properties of the coatings was also assessed. The coatings demonstrated high durability and high biocompatibility. The bias voltage and bias mode affected both the hardness and residual stress of the Si/DLC coatings. Particularly, application of 60 V negative bias during the DLC layer deposition resulted in the lowest wear rate. FEM simulations showed that the wear resistance of the coatings was dictated by the hardness as well as the adhesion between the coatings and a chromium sub-layer. The periodical alternation of Si and DLC nanolayers led to a significant improvement of MC3T3 cell adhesion compared to the previously published data for Si-DLC composites.