The effect of the combined application of urease and nitrification inhibitors on ammonia volatilization and the abundance of nitrifier and denitrifier communities is largely unknown. Here, in a mesocosm experiment, ammonia volatilization was monitored in an agricultural soil treated with urea and either or both of the urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) and the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP), with 50% and 80% water-filled pore space (WFPS). The effect of the treatments on the abundance of bacteria and archaea was estimated by quantitative PCR (qPCR) amplification of their respective 16S rRNA gene, that of nitrifiers using amoA genes, and that of denitrifiers by qPCR of the norB and nosZI denitrification genes. After application of urea, N losses due to NH 3 volatilization accounted for 23.0% and 9.2% at 50% and 80% WFPS, respectively. NBPT reduced NH 3 volatilization to 2.0% and 2.4%, whereas DMPP increased N losses by up to 36.8% and 26.0% at 50% and 80% WFPS, respectively. The combined application of NBPT and DMPP also increased NH 3 emissions, albeit to a lesser extent than DMPP alone. As compared with unfertilized control soil, both at 50% and 80% WFPS, NBPT strongly affected the abundance of bacteria and archaea, but not that of nitrifiers, and decreased that of denitrifiers at 80% WFPS. Regardless of moisture conditions, treatment with DMPP increased the abundance of denitrifiers. DMPP, both in single and in combined application with NBPT, increased the abundance of nitrification and denitrification genes.