Both, 3,4-dimethylpyrazole phosphate (DMPP) and N-(n-butyl) thiophosphoric triamide (NBPT) are commonly used as nitrogen (N) stabilizers, and are often used in agriculture to reduce nitrogen (N) loss from soils by inhibiting soil nitrification and by slowing down urea hydrolysis, respectively. The current knowledge gap concerns how soil microbial communities involved in N cycling are affected by the long-term use of DMPP and NBPT. The present field study explored the inter-annual variation of nitrous oxide (N2O) emissions and the responses of ammonia oxidizers (AOA, AOB encoded by the amoA gene), nitrite-oxidizing bacteria (NOB, encoded by the nxrA and nxrB genes), and denitrifier (encoded by the narG and nosZ genes) populations following a long-term (8 years) addition of DMPP and NBPT. The results showed that the reduction in N2O emissions by DMPP and NBPT increased year on year. The AOB population diversity significantly increased (p < 0.05) after a long-term urea application but decreased after DMPP addition. The long-term application of urea increased the potential nitrification rate (PNR) by the enrichment of the genera with a high ammonia oxidation capacity in the AOB population. In contrast, DMPP addition weakened this effect and formed a population with a low ammonia oxidation capacity. Variations in the NOB population were mainly associated with fertilizer-induced changes in substrate NO2−, whereas DMPP and NBPT had minor impacts on the NOB population. Additionally, the change in the denitrification population was indirectly affected by the soil ammonium (NH4+) content with a long-term N stabilizer application. These findings provide a new interpretation related to the response mechanisms of the nitrifier and denitrifier populations for the long-term use of N stabilizers in soils.