Recently, graphene oxide (GO)-based nanocomposite has raised significant interests in many different areas, one of which being antibacterial agents where sliver nanoparticles (AgNPs) anchored GO (GO-Ag) has shown promising potentials. However, to our best knowledge, factors affecting its antibacterial activity as well as the underlying mechanism remain unclear. In this study, we fabricate GO-Ag nanocomposites with different AgNPs to GO ratios, and carefully investigate their antibacterial activities against both the Gram-negative (G-) bacteria Escherichia coli (E. coli) and the Gram-positive (G+) bacteria Staphylococcus aureus (S. aureus). We discover that, compared to AgNPs, GO-Ag nanocomposite with an optimal ratio of AgNPs to GO is much more effective and shows synergistically enhanced, strong antibacterial activities at rather low dose (2.5 μg/mL). The GO-Ag nanocomposite is more toxic to E. coli than that to S. aureus. The antibacterial effects of GO-Ag nanocomposite are further investigated, revealing distinct, species-specific mechanisms. The results demonstrate that GO-Ag nanocomposite functions as a bactericide against the G- E. coli through disrupting bacterial cell wall integrity, whereas it exhibits bacteriostatic effect on the G+ S. aureus by dramatically inhibiting cell division. Our work not only highlight the great promise of using GO-Ag as a highly effective antibacterial agent, but also provide more in-depth understandings of the interactions between microorganisms and GO-based nanocomposites.