Caves are unique habitats and important components of their ecosystems. Caves are also vital to local economies because they serve as tourist attractions. Cave habitats appear to host diverse communities of fungi. In this study we explore associations between levels of human disturbance and the diversity of fungi in four caves in Kentucky and Tennessee. Species isolated from cave soils were cultured at 10 C (cave temperature) and room temperature. The results show that fungal diversity is low in heavily trafficked sites, increases in moderately visited sites and peaks at low disturbance levels. No fungi were cultured from sites that had very rarely or never been entered before we sampled. Species were counted using the morphological species concept. Fungi from the most diverse site were also characterized using sequence data of the ITS locus. Using this method the fungi were identified as species of Bionectria, Cadophora, Fusarium, Hypocrea, Mortierella, Paraconiothyrium, Penicillium and Podospora.