The antibacterial and barrier properties of natural rubber used as gloves are very important for the safety of medical staffs. In this research, the silver (Ag) particles were loaded on the surface of graphene oxide (GO) first modified by polydopamine (PDA). Then, the complex particles (Ag-PDA-GO) were introduced into the natural rubber (NR) latex, and the Ag-PDA-GO/NR film composites were obtained by the dipping method. Results showed that a fine dispersion of Ag-PDA-GO in NR film was obtained due to the isolation effect of Ag and PDA between GO sheets. Compared with those of pristine NR composite, when the GO content was only 0.2 phr, the tensile strength, tear strength and modulus at 100% and 300% strains of the composites increase by 66.7%, 128%, 37.7% and 30.7%, respectively, compared with the pure NR. The gas diffusion coefficient was reduced by 15.6% due to the strong interface interaction between GO and NR macromolecules. When the GO content was only 0.1 phr, the minimum inhibitory concentrations (MIC) of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were 16 and 32 mu g/mL, respectively. These results are of great significance for improving the barrier and antibacterial properties of medical rubber products.