The effects of antibiotics on bacterial infections are gradually weakened, leading to the wide development of nanoparticle-based antibacterial agents with unique physical and chemical properties and antibacterial mechanisms different from antibiotics. In this study, we fabricated the uniform and stable graphene oxide (GO)/Ni colloidal nanocrystal cluster (NCNC) nanocomposite by electrostatic self-assembly and investigated its synergistic antibacterial activity against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) in vitro. The GO/NCNC nanocomposite was shown to possess higher inhibition efficiency than a pure NCNC or GO suspension, with 99.5 and 100% inhibition against S. aureus and E. coli at a 125 μg/mL concentration, respectively. Antibacterial mechanism analysis revealed that (i) NCNCs decorated on GO can further enhance the antibacterial properties of GO by binding and capturing bacteria, (ii) the leaching of Ni2+ was detected during the interaction of GO/NCNCs and bacteria, resulting in a decrease in the number of bacteria, and (iii) the GO/NCNC nanocomposite can synergistically destroy the bacterial membrane through physical action and induce the reactive oxygen species generation, so as to further damage the cell membrane and affect ATPase, leakage of intercellular contents, and ultimately bacterial growth inhibition. Meanwhile, cell culture experiments demonstrated no adverse effect of GO/NCNCs on cell growth. These preliminary results indicate the high antibacterial efficiency of the GO/NCNC nanocomposite, suggesting the possibility to develop it into an effective antibacterial agent in the future against bacterial infections.