Food webs in Southern Appalachian forest soils are complex. Because of climate uncertainty, it is imperative that we gain a baseline understanding of the nutrient fluxes associated with each trophic level of the forest ecosystem. Microbial carbon deposition and respiration are responsible for large portions of the soil carbon flux but there is little research detailing microbial interactions with soil macrofauna. This study examines the effect of Aphaenogaster ants on soil microbial diversity. These ants participate in a keystone mutualism with a guild of myrmecochorous plants in Southern Appalachian forests. The data presented here demonstrate that Aphaenogaster interactions extend beyond the guild of herbaceous plants. Fungal diversity increases in ant nest soil relative to bulk soil (p = 0.028). Elucidating the ecology of Aphaenogaster spp. could be a key to understanding nutrition webs in the deciduous forests of eastern North America.