Abstract Continuously rising atmospheric CO₂ concentrations may lead to an increased transfer of organic C from plants to the soil through rhizodeposition and may affect the interaction between the C- and N-cycle. For instance, fumigation of soils with elevated CO₂ (eCO₂) concentrations (20% higher compared to current atmospheric concentrations) at the Giessen Free-Air Carbon Dioxide Enrichment (GiFACE) sites resulted in a more than 2-fold increase of long-term N₂O emissions and an increase in dissimilatory reduction of nitrate compared to ambient CO₂ (aCO₂). We hypothesized that the observed differences in soil functioning were based on differences in the abundance and composition of microbial communities in general and especially of those which are responsible for N-transformations in soil. We also expected eCO₂ effects on soil parameters, such as on nitrate as previously reported. To explore the impact of long-term eCO₂ on soil microbial communities, we applied a molecular approach (qPCR, T-RFLP, and 454 pyrosequencing). Microbial groups were analyzed in soil of three sets of two FACE plots (three replicate samples from each plot), which were fumigated with eCO₂ and aCO₂, respectively. N-fixers, denitrifiers, archaeal and bacterial ammonia oxidizers, and dissimilatory nitrate reducers producing ammonia were targeted by analysis of functional marker genes, and the overall archaeal community by 16S rRNA genes. Remarkably, soil parameters as well as the abundance and composition of microbial communities in the top soil under eCO₂ differed only slightly from soil under aCO₂. Wherever differences in microbial community abundance and composition were detected, they were not linked to CO₂ level but rather determined by differences in soil parameters (e.g., soil moisture content) due to the localization of the GiFACE sets in the experimental field. We concluded that +20% eCO₂ had little to no effect on the overall microbial community involved in N-cycling in the soil but that spatial heterogeneity over extended periods had shaped microbial communities at particular sites in the field. Hence, microbial community composition and abundance alone cannot explain the functional differences leading to higher N₂O emissions under eCO₂ and future studies should aim at exploring the active members of the soil microbial community.