How nitrogen (N) deposition impacts ectomycorrhizal (EM) fungal communities has been little studied in deciduous forests or across spatial scales. Here, it was tested whether N addition decreases species richness and shifts species composition across spatial scales in temperate deciduous oak forests. Combined molecular (terminal restriction fragment length polymorphism (T-RFLP), sequencing) and morphological approaches were used to measure EM fungal operational taxon unit (OTU) richness, community structure and composition at the spatial scale of the root, soil core and forest during a 3-yr N fertilization experiment in Quercus-dominated forests near Chicago, IL, USA. In N treatments, significantly lower OTU richness at the largest but not smaller spatial scales and a different community structure were detected. The effects of N appeared to be immediate, not cumulative. Ordination indicated the composition of EM fungal communities was determined by forest site and N fertilization. The EM fungi responded to a N increase that was low compared with other fertilization studies, suggesting that moderate increases in N deposition can affect EM fungal communities at larger spatial scales in temperate deciduous ecosystems. While responses at large spatial scales indicate that environmental factors can drive changes in these communities, untangling the impacts of abiotic from biotic factors remain limited by detection issues.