The antimicrobial effects of silver (Ag) ion or salts are well known, but the effects of Ag nanoparticles on microorganisms and antimicrobial mechanism have not been revealed clearly. Stable Ag nanoparticles were prepared and their shape and size distribution characterized by particle characterizer and transmission electron microscopic study. The antimicrobial activity of Ag nanoparticles was investigated against yeast, Escherichia coli, and Staphylococcus aureus. In these tests, Muller Hinton agar plates were used and Ag nanoparticles of various concentrations were supplemented in liquid systems. As results, yeast and E. coli were inhibited at the low concentration of Ag nanoparticles, whereas the growth-inhibitory effects on S. aureus were mild. The free-radical generation effect of Ag nanoparticles on microbial growth inhibition was investigated by electron spin resonance spectroscopy. These results suggest that Ag nanoparticles can be used as effective growth inhibitors in various microorganisms, making them applicable to diverse medical devices and antimicrobial control systems.