A natural carbon dioxide (CO2) vent sited in Ciudad Real (Spain) was studied to understand how CO2 emissions affect microbial communities along a CO2 gradient. We used different molecular methods (quantitative PCR, DGGE and Biolog EcoPlates™) to assess changes in abundance, diversity and functionality of the main groups of soil microbiota (bacteria, archaea and fungi). A general decrease for all studied variables (gene copies and band richness of bacteria, archaea and fungi, and Biolog activities) was observed from control (7–19gm−2d−1) to high CO2 fluxes (260–1600gm−2d−1). On the contrary, at extreme fluxes (more than 10kgm−2d−1) the microbial community increased their abundance and activity, though remained less diverse. PCA from carbon use substrate pattern and DGGE dendograms clearly differentiated low fluxes from high and extreme. This paper proves that high CO2 fluxes (between 260 and 1600gm−2d−1) cause losses in both structural and functional community diversity, and a decrease in metabolic activities.