Silver nanoparticles (AgNPs) is widely used as an antibacterial agent, but the specific antibacterial mechanism is still conflicting. This study aimed to investigate the size dependent inhibition of AgNPs and the relationship between inhibition and reactive oxygen species (ROS). Azotobactervinelandii and Nitrosomonaseuropaea were exposed to AgNPs with different particles size (10 nm and 50 nm). The ROS production was measured and the results showed that the generation of ROS related to the particle size and concentrations of AgNPs. At 10 mg/L of 10 nm Ag particles, the apoptosis rate of A. vinelandii and N. europaea were 20.23% and 1.87% respectively. Additionally, the necrosis rate of A. vinelandii and N. europaea reached to 15.20% and 42.20% respectively. Furthermore, transmission electron microscopy images also indicated that AgNPs caused severely bacterial cell membrane damage. Together these data suggested that the toxicity of AgNPs depends on its particle size and overproduction of ROS.