These data comprise arbuscular mycorrhizal (AM) fungi diversity data from Sourhope field experiment site. AM fungi are biotrophic symbionts colonizing the majority of land plants, and are of major importance in plant nutrient supply. Using the terminal restriction fragment length polymorphism (T-RFLP) strategy, the diversity of AM fungi was assessed in 89 roots of three grass species (Agrostis capillaris, Festuca rubra, Poa pratensis) that co-occurred in the same plots of the Sourhope field experiment. The impact of different soil amendments (nitrogen, lime, nitrogen and lime) and insecticide application on AM fungal community was also recorded. Data were collected during a project funded under the NERC Soil Biodiversity Programme, established in 1999 and centred upon the intensive study of a large field experiment located at the Macaulay Land Use Research Institute (now the James Hutton Institute) farm at Sourhope in the Scottish Borders (Grid reference: NT 8545 1963). During the experiment, the site was monitored to assess changes in above-ground biomass production (productivity), species composition and relative abundance (diversity). Full details about this dataset can be found at https://doi.org/10.5285/5aa0e9ee-9604-4cba-a1ae-5d6708cf6438