Macrofungi from two old-growth prairie grove remnants in the Midwestern United States (Brownfield and Trelease Woods, Champaign Co., IL ) were surveyed over two summer and fall fruiting periods. Communities of Ascomycetes, Basidiomycetes and Myxomycetes were sampled and compared using multivariate statistical analyses. Standard estimations of species richness were calculated for comparison with other studies of fungal diversity. Environmental factors (rainfall, humidity, air temperature and soil temperature at 10 cm depth) as well as leaf litter composition, and woody plant communities were surveyed to assess their impact on fungal communities. Fungal community structure was found to differ significantly both between and within Brownfield and Trelease Woods. Communities of terrestrial macrofungi were determined to be strongly influenced by seasonality, with soil temperature at 10 cm depth showing the strongest correlation to changes in community composition. Brownfield and Trelease Woods, formerly part of a contiguous prairie grove with likely a single fungal community, are shown to have developed significantly different fungal communities over a period of separation of more than 120 y.