In this paper, the influence of material composition on structure and surface properties of bioactive coatings based on Cu and Ti is described. Nanocrystalline coatings were prepared by innovative pulsed DC magnetron sputtering. For their preparation, a multi-magnetron system was used in order to obtain films with various copper content. The main goal of our work was the complex analysis of biological activity of Cu-Ti films in comparison with their material composition and surface state. Antimicrobial activity (for E. coli and S. aureus), as well as the impact on cell viability (L929 line), were investigated. The physicochemical properties were examined with the aid of X-ray diffraction, scanning electron microscopy, X-ray photoelectron spectroscopy, and atomic absorption spectroscopy. It was found that all prepared films were nanocrystalline and bactericidal, but their cytotoxicity was related to the Cu-content in the film. Complex analysis of the bioactivity was developed in relation to the copper ion migration process. Moreover, manufacturing of antibacterial films with stimulating action on L929 cell line was possible.