Plots containing Lolium perenne L., Trifolium repens L. or a mixture of both plant species were exposed to elevated atmospheric CO2 (eCO2) for 10 consecutive seasons using free-air CO2 enrichment technology at ETH Zürich, Switzerland. The CO2 treatment was crossed with a two-level nitrogen (N) fertilization treatment. In the tenth year, soil samples were collected on three occasions through the growing season to assess the impact of eCO2 and N fertilization on mycorrhizal fungal abundance. Soil moisture content, which varied with harvest date, was linked to the vegetation type and was higher under eCO2. Root weight density was affected by vegetation type: lower for clover, higher for grass. Root weight density was stimulated by eCO2 and decreased by high N fertilization. The percent root length colonized by mycorrhizal fungi was lowest in the clover plots and highest in the grass plots. High N significantly decreased root length colonized. There was no overall effect of eCO2 on root length colonized; however, there was a significant eCO2× N interaction: eCO2 increased root length colonized at high N, but decreased root length colonized at low N. Extraradical mycorrhizal hyphal density was linked to soil moisture content. Extraradical mycorrhizal hyphal density was not affected by eCO2 or high N individually, but as for root length colonized, there was a significant eCO2× N interaction: eCO2 increased extraradical mycorrhizal hyphal density at low N but not at high N. These environmental effects on root colonization and external mycorrhizal hyphae were independent of soil moisture content and root weight density. This field study demonstrated a significant mediating effect of N fertilization on the response of arbuscular mycorrhizal fungi to eCO2 irrespective of any change in root biomass.