The occurrence of antibiotic resistance against pathogens is rapidly increasing and endangering the efficacy of antibiotics. Thus, finding a way to address this problem has become a major challenge due to the inability of conventional antibiotics to kill these multidrug-resistant bacteria. In order to further enhance the antibacterial ability and reduce the possibility of antibiotic resistance, we developed a simple two-step approach and synthesized a new nanocomposite by directly loading single-stranded DNA (ssDNA)-guided silver nanoparticles (AgNPs) on graphene oxide (ssDNA-AgNPs@GO). Through systematically evaluating the bactericidal activity and wound healing capability, we found that ssDNA-AgNPs@GO exhibited synergistic antibacterial activity against , , and with low minimum inhibitory concentrations (6.8 μg mL, 6.8 μg mL, 11.9 μg mL and 10.2 μg mL, respectively) and large-diameter inhibition zones (12.83 ± 0.63 mm, 13.14 ± 0.37 mm, 8.6 ± 0.9 mm and 8.93 ± 0.47 mm, respectively). Furthermore, the wound healing experiment indicated that it has a striking ability to remedy wound infection caused by bacteria. In conclusion, the properties of ssDNA-AgNPs@GO with enhanced antibacterial and wound healing capability will give it broad applications in the future.