As part of a broad multidisciplinary investigation to elucidate the role of chemical speciation in biological efficacy, novel silver films exhibiting antimicrobial behaviour were produced by magnetron sputtering a pure silver cathode. These physically vapour-deposited films, grown using a range of gas compositions, gas pressures and input powers, were studied in situ and ex situ using X-ray photoelectron spectroscopy (XPS), in both the as-deposited and the annealed condition. In the in-situ case, a special system was employed to transfer the sample to the analytical chamber of the XPS without exposure to the ambient environment. On the basis of the data obtained, it is postulated that oxygen species detected in the films are present in several chemical states. Furthermore, silver films deposited under specific process conditions exhibited antimicrobial behaviour in laboratory biological tests. The unique antimicrobial properties are explained in terms of the bulk composition, microchemistry and chemical state of the constituents present, as well as the microstructural features of the physically vapour-deposited silver films.