Natural biological suppression of soil-borne diseases is a function of the activity and composition of soil microbial communities. The incidence of soil-borne disease depends on interactions between phyto-pathogenic fungi and endemic bacterial and fungal communities. These interactions can occur prior to crop sowing and/or in the rhizosphere, subsequently influencing both plant growth and productivity. Research on suppressive microbial communities has concentrated on bacteria although fungi can also influence soilborne disease impacts. We analyzed fungi in co-located ?suppressive? and ?non-suppressive? soils for disease caused by Rhizoctonia solani AG 8 at two sites in South Australia using 454 pyrosequencing targeting the fungal 28S LSU rRNA gene. Using a minimum of 125 g of soil for DNA extraction reduced the micro-scale variability in the distribution of soil fungi. We obtained >1 million reads classified into 917 genera covering 54% of the RDP Fungal Classifier database, a high diversity for an alkaline, low organic matter soil. Approximately 33% of the fungal community remained unclassified at the genus level using a 50% bootstrap classification confidence. Statistical analyses and community ordinations revealed significant differences between suppressive and non-suppressive soils and between soil type/location. The majority of differences associated with suppressive soils were attributed to less than 40 genera including a number of endophytic species with plant pathogen suppression potentials and mycoparasites, e.g. Xylaria spp. while non-suppressive soils were dominated by Alternaria, Gibberella, and Penicillum. Pyrosequencing generated a detailed description of fungal community structure and emphasized the importance of fungal communities that may influence pathogen-plant interactions in stable disease suppression.