Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are considered as the key drivers of global nitrogen (N) biogeochemical cycling. Responses of the associated microorganisms to global changes remain unclear. This study was to determine if there was a shift in soil AOB and AOA abundances and community structures under free-air carbon dioxide (CO2) enrichment (FACE) and N fertilization in Duke Forest of North Carolina, by using DNA-based molecular techniques, i.e., quantitative PCR, restriction fragment length polymorphism (RFLP) and clone library. The N fertilization alone increased the abundance of bacterial amoA gene, but this effect was not observed under elevated CO2 condition. There was no significant effect of the N fertilization on the thaumarchaeal amoA gene abundance in the ambient CO2 treatments, while such effect increased significantly under elevated CO2. A total of 690 positive clones for AOA and 607 for AOB were selected for RFLP analysis. Analysis of molecular variance (AMOVA) indicated that effects of CO2 enrichment and N fertilization on the community structure of AOA and AOB were not significant. Canonical correspondence analysis also showed that soil pH rather than elevated CO2 or N fertilization shaped the distribution of AOB and AOA genotypes. A negative linear relationship between the d13C and archaeal amoA gene abundance indicated a positive effect of elevated CO2 on the growth ammonia oxidizing archaea. On the other hand, the community structures of AOB and AOA are determined by the soil niche properties rather than elevated CO2 and N fertilization.