In 2004, we determined baseline data on fungal-community assemblages from Solenopsis invicta (Red Imported Fire Ant) mounds in 3 counties (Hinds, Leake, and Madison) within the Natchez Trace Parkway, MS. We assayed mound soil, plant debris within the mounds, and ants obtained from mounds on 3 sampling dates (March, July, and November). We processed samples based on standard microbiological protocols, and used traditional morphological and molecular techniques to identify fungal taxa. We documented a total of 1445 isolates consisting of 50 fungal taxa and calculated a diversity index value (H′) of 3.11 across all substrates, which was indicative of a variable fungal community within the mounds. The taxa with the highest percent isolation frequencies included Hypocrea lixii (12.8%), Fusarium sp. 1 (12.3%), Fusarium equiseti (7.9%), Purpureocillium lilacinum (= Paecilomyces lilacinus) (6.5%), Fusarium oxysporum 2 (5.8%), and Mortierella alpina (5.4%). We isolated 2 common parasitic (entomopathogenic) fungi, Purpureocillium lilacinum and Metarhizium anisopliae var. anisopliae (9.4%), from mound soil, plant debris, and ant external tissues. Hypocrea lixii, the teleomorphic reproductive stage of Trichoderma harzianum, is noted as a natural biological control of some soil-borne microbes, possibly limiting important natural entomopathogenic activity within the mounds. Species richness and diversity values from mound soils across locations were significantly greater (P ≤ 0.05) than those from the plant debris and ant body-tissue substrates. Species richness values between locations were similar. Species richness of samples collected in November (47) was significantly greater (P ≤ 0.05) than that of the March (41) and July (39) samples. Community coefficient values ranged from 0.79 to 0.87 between substrates, 0.85 to 0.91 between locations, and 0.85 to 0.86 between sampling dates, indicating that taxa were similar.