Graphene and silver nanoparticles (AgNPs) are important building blocks for the synthesis of functional nanomaterials for bio-related applications. Here, we report a facile strategy to decorate AgNPs onto reduced graphene oxide (RGO) by the simultaneous reduction of silver ions and graphene oxide nanosheets within one system, and further to fabricate a dimension-adjustable RGO/AgNP multi-layered film by a thermal-driven self-assembly process. The structures of the fabricated RGO/AgNP hybrid films were identified by UV-visible spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, Raman spectroscopy, and transmission electron microscopy. The thickness of the fabricated RGO/AgNP film was further measured by scanning electron microscopy. The hydrophilicity of the RGO/AgNP films was tested by contact angle measurement. Antibacterial and cell culture experiments based on the fabricated RGO/AgNP films indicate that this kind of hybrid film exhibits excellent antibacterial activity and high biocompatibility. A potential antibacterial mechanism of the fabricated RGO/AgNP hybrid film was proposed.