Prior to the loss of Tsuga canadensis (Eastern Hemlock) stands in the Great Smoky Mountains National Park (GRSM), we collected baseline data during 2006–2009 at two locations (Copeland Creek and Gabes Mountain) regarding macrofungi that occur under this tree species. We studied macrofungi in order to understand the current and changing ectomycorrhizal and saprobic fungal community structure associated with healthy (imidicloprid-treated) and dying Eastern Hemlock stands and to contribute data for the All Taxa Biodiversity Inventory (ATBI) in GRSM. A total of 121 taxa representing 75 ectomycorrhizal, 1 pathogenic, and 45 saprobic species, were collected from 92, 59, and 106 sampling locations in 2006, 2007, and 2008, respectively. Macrofungal species richness, diversity, and evenness (E) in Copeland Creek were significantly greater (P = 0.05) in 2006 than 2007. Eighty percent of all fungi collected were found in Copeland Creek (487 m elevation) where trees are <75 years old; the remaining 20% were collected at Gabes Mountain (1158 m elevation) where trees are >150 years old. The most common taxa found across sampling locations included Russula fragrantissima (22.0%), Amanita citrina var. lavendula (Lavender False Death Cap; 17.1%), Austroboletus gracilis (Graceful Bolete; 14.6%), Laccaria laccata (Deceiver; 9.8%), and Russula russuloides (9.8%). Amanita cinereopannosa occurred at a low frequency overall (2.4% of trees sampled) but when present had high abundance. Total macrofungi and ectomycorrhizal fungi at Copeland Creek had significantly greater species richness, density, and evenness than at the Gabes Mountain site. Ectomycorrhizal fungi evenness values following imidicloprid soil-drenching treatments were significantly less uniform for macrofungi collected from within 5 m of trees treated in subplots with full rates of the chemical than for those near untreated trees (E = 0.2 and 0.4, respectively). In addition, there was a numerical trend towards significantly less diversity and evenness at the full chemical application rate compared to the half rate or untreated control plots. There were no differences in the occurrence of saprobic fungi between chemically treated and control trees. Associated vegetation had a significant impact on the occurrence of macrofungi. Across both locations, a total of 37 plant, shrub, or tree species were identified; Acer pensylvanicum (Striped Maple) with an overall abundance of 11.0%, Pyrularia pubera (Buffalo Nut; 10.5%), Ilex opaca (American Holly; 8.2%), Calycanthus floridus (Eastern Sweetshrub; 4.8%), and Rhododendron maximum (Great Rhododendron; 4.7%), were the most common associated species. Species richness, diversity, and evenness of the associated vegetation were significantly different between locations. Evenness data for plant species abundance was not equal, but varied within and across locations.