A study was conducted to determine microbial community structure and baseline information of cultural bacteria taxa within Solenopsis invicta (Red Imported Fire Ant) mounds from 3 locations along the roadside of Natchez Trace Parkway in Mississippi. At each location, samples consisting of mound soils, plant debris of primarily grass stem and leaves (control), and ant body tissues were obtained from replicate mounds during March, July, and November 2004. Bacteria isolate frequencies from soil were significantly greater than from plant or ant body tissues. Using 16S sequence data, 68 taxa from 2324 isolates were obtained from the 3 substrate types. The 7 most common bacteria following in order of greatest isolation frequencies were Bacillus sp. (5) (species complex), Achromobacter xylosoxidans, Bacillus cereus (complex), Lysininibacillus boronitolerans, Serratia liquefaciens, Pseudomonas protegens, and Lysinibacillus sphaericus. Richness, diversity, and evenness values varied between the locations, sampling dates, and the 3 isolation substrates. Total community-coefficient values were 0.74 to 0.84 across sampling dates. Overall these values indicated uniform communities across the different locations, isolation substrates, and across 3 sampling dates. Furthermore, no consistent trends in frequencies were observed by comparing ant tissues, location, and sampling dates to occurrences of bacterial taxa. Isolates and data obtained from this survey will allow for further testing to determine their role as food sources, saprophytes, or pathogens in Red Imported Fire Ant mound ecosystems.