In this study, the physicochemical and surface properties of the GO–Ag composite promote a synergistic antibacterial effect towards both Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. Aureus) bacteria. GO–Ag NPs have a better bactericidal effect on E. coli (73%) and S. Aureus (98.5%) than pristine samples (pure Ag or GO). Transmission electron microscopy (TEM) confirms that the GO layers folded entire bacteria by attaching to the membrane through functional groups, while the Ag NPs penetrated the inner cell, thus damaging the cell membrane and leading to cell death. Cyclic voltammetry (CV) tests showed significant redox activity in GO–Ag NPs, enabling good catalytic performance towards H2O2 reduction. Strong reactive oxygen species (ROS) in GO–Ag NPs suggests that ROS might be associated with bactericidal activity. Therefore, the synergy between the physicochemical effect and ROS production of this material is proposed as the mechanism of its antibacterial activity.