Biofouling of materials has caused significant loss to marine engineering and protective coatings have served as a solution. In this work, the TiSiN/Ag multilayer coatings synthesized by ion plating were investigated. Discontinuous Ag layers in the coating was prepared to keep excellent mechanical properties of the coating. In microcosmic, it was observed that Ag ions released from the coating by ‘micro-channel’ and expressed anti-bacterial activity in seawater environment. The released rate is found to be determined by the microstructure of the coating. In order to prolong the service life of the coating, it is expected that the Ag ions release concentration can approach the minimal inhibitory concentration (MIC). In this work, the TiSiN/Ag multilayer coating can release Ag ions at a pretty low concentration and maintain its excellent biocidal activity. This work has provided a new thought to synthesize a long-lasting anti-bacterial and superhard coating.