Developing methods to differentiate the relative contributions of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) to ammonia (NH3) oxidation has been challenging due to the lack of compounds that selectively inhibit AOA. In this study, we investigated the effects of specific bacteria- and eukaryote-selective protein synthesis inhibitors on the recovery of acetylene (C2H2)-inactivated NH3 oxidation in the marine AOA Nitrosopumilus maritimus and compared the results with recovery of the AOB Nitrosomonas europaea. C2H2 irreversibly inhibited N. maritimus NH3 oxidation in a similar manner to what was observed previously with N. europaea. However, cycloheximide (CHX), a widely used eukaryotic protein synthesis inhibitor, but not bacteria-specific protein synthesis inhibitors (kanamycin and gentamycin), inhibited the recovery of NH3-oxidizing activity in N. maritimus. CHX prevented the incorporation of 14CO2-labeling into cellular proteins, providing further evidence that CHX acts as a protein synthesis inhibitor in N. maritimus. If the effect of CHX on protein synthesis can be confirmed among other isolates of AOA, the combination of C2H2 inactivation followed by recovery of NH3 oxidation either in the presence of bacteria-selective protein synthesis inhibitors or CHX might be used to estimate the relative contributions of AOB and AOA to NH3 oxidation in natural environments.