As part of an All Taxa Biodiversity Inventory of the Great Smoky Mountains National Park, an assemblage of microfungi associated in litter samples from healthy Fagus grandifolia (American Beech), Abies fraseri (Fraser Fir), and Tsuga canadensis (Eastern Hemlock) trees was determined in 2005 and 2006. Additionally, litter samples from the collection sites were assayed for pH, nutrient content, ash, crude proteins, and levels of organic matter to determine their impact on the mycobiota. Species richness, diversity, and evenness patterns were evaluated from the litter samples collected in May, July, and September of each year. A total of 6249 isolates of fungi were obtained, with greater than 90% belonging to the Deuteromycota. Over 100 species of fungi were identified from litter of the three tree species, with 55 being new records from the Park. As in previous studies, the most common fungi isolated from the three tree species were 13 species of Trichoderma during the two-year study. Other common fungi included Virgaria nigra and Penicillium spp. Species richness and diversity values pooled across sampling dates and years were significantly greater from American Beech litter, followed by Eastern Hemlock and lowest for Fraser Fir. Species richness and diversity values compared by sampling dates for each year were generally greater in May than July or September, but evenness values showed a reverse trend for each year. When species richness and diversity were compared between sampling dates per year and among or by tree species, significant differences often occurred, but no trends were determined. Data from the litter tissue assay showed that Fraser Fir, which had the lowest species richness and diversity, may have been impacted by having significantly lower pH and percent litter chemical compositions of ash, crude protein, and N than the other tree species. All other comparisons of species richness were similar.