The newly-discovered complete nitrification process by “comammox” bacteria, capable of oxidizing ammonium to nitrate in a single organism, is hypothesized to play a crucial role in the terrestrial nitrogen cycle. However, the responses of comammox bacteria to nitrification inhibitors and their relative contribution to nitrification in agricultural soils remain largely unknown. Here, we carried out a microcosm study to evaluate the impacts of four different nitrification inhibitors (acetylene, 1-octyne, nitrapyrin and DMPP) and N fertilizer ((NH4)2SO4) on the abundance of comammox Nitrospira, ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in pasture and vegetable soils. We found that addition of (NH4)2SO4 significantly increased the nitrate concentrations and addition of all these four nitrification inhibitors effectively reduced the production of nitrate in soil. Furthermore, the amoA gene abundances of comammox Nitrospira clade A, AOA and AOB significantly increased after the application of (NH4)2SO4. The amoA gene abundances of comammox clade A were significantly inhibited by acetylene and nitrapyrin. AOB was significantly inhibited by all these four nitrification inhibitors though to varying degrees, and AOA was significantly inhibited only by acetylene. These results indicated acetylene could effectively impede the abundance of comammox Nitrospira and canonical nitrifiers, but 1-octyne only could inhibit the growth of AOB in agricultural soils. Our findings will provide valuable scientific basis for the efficiency of different nitrification inhibitors on the growth of comammox Nitrospira and canonical nitrifiers in agricultural management practices.