The bio-toxicity of Fe0 nanoparticles on the denitrifying bacterial- Alcaligenes eutrophus was detected by the two methods of detecting inhibition of the growth of microorganisms and nitrification inhibition rate. The results showed that Fe0 nanoparticles had obvious toxicity on the growth and nitrification inhibition rate. Besides, there had distinct relationship between dose and toxicity of Fe0 nanoparticles. Meanwhile, we also studied effects of different kinds of Fe0 nanoparticles on microorganism, including bimetallic nanoparticles(nano Fe–Ni) and coated iron nanoparticles (sodium oleate–Fe0nanoparticles). We detected that the bio-toxicity of these three Fe0 nanoparticles followed the sequence of Ni-containing nanoparticles>unmodified NZVI>the sodium oleate–Fe0 nanoparticles. In conclusion, Fe0 nanoparticles had visible toxicity on the denitrifying bacterial, but we can decrease the toxicity of Fe nanoparticles by surface coating and decoration of nano-metal materials.