Nitrogen (N) availability is increasing in many ecosystems due to anthropogenic disturbance. We used a nucleotide analog technique and sequencing of ribosomal RNA genes to test whether N fertilization altered active fungal communities in two boreal ecosystems. In decaying litter from a recently burned spruce forest, Shannon diversity decreased significantly with N fertilization, and taxonomic richness declined from 44 to 33 operational taxonomic units (OTUs). In soils from a mature spruce forest, richness also declined with N fertilization, from 67 to 52 OTUs. Fungal community structure in litter differed significantly with N fertilization, primarily because fungi of the order Ceratobasidiales increased in abundance. We observed similar changes in fungal diversity and community structure with starch addition to litter, suggesting that N fertilization may affect fungal communities by altering plant carbon inputs. These changes could have important consequences for ecosystem processes such as decomposition and nutrient mineralization.