Long-term increases in nitrogen (N) availability resulting from anthropogenic N deposition can strongly influence ectomycorrhizal (EM) fungal communities with potential consequences for nutrient cycling, forest composition and carbon storage. In a tropical montane forest in Panama, we used Illumina amplicon sequencing to examine how 9 y of experimental N addition has affected EM communities associated with Oreomunnea mexicana, and fluorescence assays to measure changes in enzyme activity in the soil. Nitrogen addition significantly reduced EM colonization of Oreomunnea roots and altered EM composition. The abundance of Laccaria and Lactarius increased with N addition, while Cortinarius declined. In addition, we found a reduction in soil phosphatase, N-acetyl-glucosaminidase, and β-xylanase activity with N addition. We conclude that a reduction in EM fungal taxa specialized in organic N and phosphorus absorption along with a decrease in EM colonization of host plants could decrease soil enzyme activity and therefore have feedback effects on soil nutrient cycling.