Investigation of the microstructure, properties and biocompatibility of the Ti-6Al-4V alloy nitrided under glow discharge was performed. The microstructural analyses were carried out using light microscopy, X-ray diffraction, analytical scanning and transmission electron microscopy. Phase identifications and chemical composition of the layer and bulk material (substrate) were determined by electron diffraction and energy dispersive X-ray spectrometry. Atomic force microscopy was applied for layer surface topography measurements. Microhardness and Young’s modulus measurements as well as frictional wear resistance and corrosion resistance tests were performed. The investigation revealed a clear correlation between the micro/nanostructure and surface topography of the layer with its micromechanical, tribological and corrosion properties. In-vitro examinations of biofilm and cell behaviour show that the nitrided Ti-6Al-4V alloy exhibits good biocompatibility.