Polydopamine (PDA) functionalized silver nanoparticles (PDA-nanoAg) were synthesized by one-step method with PDA as the reductant and protectant. The mechanism for preparation of PDA-nanoAg was proposed. The stable dispersion duration of the as-synthesized Ag nanoparticles in water was also observed. The structure and morphology of PDA-nanoAg particles were investigated by ultraviolet-visible (UV-Vis) absorption spectroscopy, transmission electron microscopy (TEM), and Fourier-transform infrared (FTIR) spectrometry. The antibacterial activity of PDA-nanoAg nanoparticles against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were investigated by broth dilution. The results indicated that the average diameter of the Ag nanoparticles was 50 nm, and PDA-nanoAg particles exhibited good stability in water over a long period of time. The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) of the as-synthesized Ag nanoparticles against E. coli and S. aureus were 7.56 mg/L and 30.24 mg/L, respectively.