To obtain a self-disinfecting surface to combat nosocomial infections, in situ synthesis of the MoO3-SiO2-Ag2O nanocomposite antibacterial coating was prepared onto Ti–6Al–4V substrate via a double cathode glow discharge technique conducted in a Ar + O2 gas mixture. The phase composition, microstructure and chemical structure of the as-prepared coating were systematically characterized by means of a series of microscopic examination methods. The microstructure of the coating exhibited an amorphous/nanocrystalline architecture, and both the MoO3 and Ag2O crystallites were evenly embedded in an amorphous SiO2 matrix. Photocatalytic activities of the coating were evaluated by photodegradation of rhodamine B (RhB) dye. The results showed that the introduction of Ag2O enhanced photocatalytic activity of the MoO3-SiO2 nanocomposite coating. In vitro antibacterial activity of the MoO3-SiO2-Ag2O coating against Gram-negative bacteria, Gram-positive bacteria and fungi were investigated and compared with that of the MoO3-SiO2 coating under both dark and visible light conditions. The MoO3-SiO2-Ag2O coating possessed higher bactericidal activities than the MoO3-SiO2 coating, due to the combined effects of surface hydrophobicity, the release of Ag+ ions, surface acidic reaction and photocatalytic activity.