Mycorrhizal fungi form ecologically important connections between plants and soils, and although nitrogen (N) enrichment has been implicated in the decline of ectomycorrhizal fungal diversity, they are rarely considered in studies investigating the effects of increased N availability on plant species diversity. This chapter describes the effects of N enrichment on the soil fungal community and in particular on arbuscular mycorrhizal fungal (AMF) spores, in a Mediterranean ecosystem in a Natura 2000 site in southern Portugal (PTCON0010 Arrábida/Espichel). Soil fungal community structure was affected by the addition of 80 kg N ha−1 year−1 as NH4NO3 within 2 years. The effects of N addition on AMF diversity (richness and evenness) appear to depend on the form of N, since the addition of 40 kg N ha−1 year −1 as ammonium increased AMF spore richness and evenness proportionally more than the addition of 40 kg N ha−1 year−1 as ammonium plus nitrate. The composition of AMF species may serve as a sensitive indicator of N enrichment.