Specific nitrification inhibitors (NIs) have been widely used to disentangle the contribution of ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB) as well as nitrite oxidizers to the nitrification process in specific environments. However, if these previously reported NIs can also be used to evaluate the activity of the newly discovered complete ammonia oxidizers (comammox) Nitrospira, remains understudied. Here we evaluated various NIs for their impact and specificity regarding inhibition of comammox Nitrospira in batch cultures of pure and mixed strains of AOA, AOB and comammox. Using these cultures, we observed that chlorate could specifically inhibit the ammonia oxidation and nitrite oxidation activity of comammox Nitrospira, while it had no effect on the tested AOA and AOB strains. This inhibitory effect of chlorate on comammox Nitrospira was subsequently confirmed based on 13CO2-DNA-stable isotope probing (13C-DNA-SIP) analysis. Furthermore, by applying a set of specific NIs, the nitrification and nitrous oxide (N2O) production rates of comammox Nitrospira in coastal wetlands were estimated as 17.45 ng N g−1 h−1 (26.9% of the total rate) and 0.0083 μmol−1 L h−1 (28.5%). Altogether, we identified and applied an effective and specific inhibitor of comammox Nitrospira, which allowed quantifying comammox activity in wetlands of the Yangtze Estuary, shedding new light on the ecological roles of comammox bacteria in coastal wetland environments.