In this work, silver nanoparticles (Ag NPs) were decorated on thiol (–SH) grafted graphene oxide (GO) layers to investigate the antibacterial activities in Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa). The quasi-spherical, nano-sized Ag NPs were attached to the GO surface layers, as confirmed by using field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM), respectively. The average size of GO-Ag nanocomposites was significantly reduced (327 nm) from those of pristine GO (962 nm) while the average size of loaded Ag NPs was significantly smaller than the Ag NPs without GO. Various concentrations of AgNO3 solutions (0.1, 0.2, and 0.25 M) were loaded into GO nanosheets and resulted in the Ag contents of 31, 43, and 65%, respectively, with 1–2 nm sizes of Ag NPs anchored on the GO layers. These GO-Ag samples have negative surface charges but the GO-Ag 0.2 M sample (43% Ag) demonstrated the highest antibacterial efficiency. At 10 ppm load of GO-Ag suspension, only a GO-Ag 0.2 M sample yielded slight bacterial inhibition (5.79–7.82%). As the GO-Ag content was doubled to 20 ppm, the GO-Ag 0.2 M composite exhibited ~49% inhibition. When the GO-Ag 0.2 M composite level was raised to 100 ppm, almost 100% inhibition efficiencies were found on both Staphylococcus aureus (S.A.) and Pseudomonas aeruginosa (P.A.), which were significantly higher than using pristine GO (27% and 33% for S.A. and P.A.). The combined effect of GO and Ag nanoparticles demonstrate efficient antibacterial activities.