Silver nanoparticles (AgNPs) with different sizes (5, 15 and 55 nm) were synthesized via simple method, and characterized by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), energy-dispersive X-ray microanalysis (EDX) and ultraviolet-visible absorption spectroscopy (UV-Vis). The antibacterial activities of the prepared AgNPs against Gram-negative Escherichia coli (E. coli), Gram-positive Staphylococcus aureus (S. aureus) and Bacillus subtilis (B. subtilis) were evaluated by inhibition zone, inhibition curve, and colony counting methods. The results showed that the AgNPs exhibited obvious bacterium-selective and size-dependent antibacterial activities. The Gram-positive bacteria S. aureus and B. subtilis were more sensitive to AgNPs than Gram-negative bacterium E. coli. Interestingly, AgNPs displayed remarkably antibacterial activities against B. subtilis among Gram-positive bacteria, regardless of whether in separately or cocultured bacteria. It also showed that AgNPs with 5 nm in size presented the highest antibacterial activity against both Gram-negative and Gram-positive bacteria. The effects of AgNPs on the membrane leakage of the reducing sugars from three bacteria were also measured by 3,5-dinitrosalicylic acid method. The leakage amount of reducing sugars from B. subtilis was the highest among the tested bacteria, indicating that AgNPs could damage the structure of bacteria cell membrane and resulted in the leakage of reducing sugars, leading to the death of bacteria.