Diuris fragrantissima is a critically endangered orchid (EPBC act 1999) native to Victoria (Australia), which grows in association with a narrow taxonomic range of mycorrhizal fungi (Tulasnella spp.) within the cosmopolitan family Tulasnellaceae (Smith et al. 2010). The limited survival of D. fragrantissima cultivated plants after reintroduction has been correlated with the loss of associated fungi during the transition from ex situ to in situ. The presence of microsites containing suitable mycorrhizal fungi able to assist the reintroduced orchids is predicted to be a necessary prerequisite to ensure the plants long-term persistence and survival in the wild (Smith et al. 2010). Monitoring the presence of Tulasnella spp. using traditional ‘baiting’ techniques had proven to be unreliable (Smith 2006). Therefore, to resolve the patterns of distribution of Tulasnella spp., we implemented a meta-barcoding approach targeting the fungal ITS region. The meta-barcoding was tested on several soil samples, which included the D. fragrantissima rhizosphere and the bulk soil from the native site. The quality-filtered data set comprised a total of 2,707 OTUs. Fungi from bulk and orchid-associated soil exhibited differing patterns of abundance at the phylum level, with a 2-fold increased richness of Basidiomycetous fungi in the rhizosphere. Overall, Tulasnella OTUs were recovered from the rhizosphere exclusively, suggesting a limited occurrence of the fungus in the Diuris native site. In addition to Tulasnella, a high diversity of OTUs associated with potentially endophytic and mycorrhizal fungi were retrieved from the orchid-associated soil, indicating that naturally occurring D. fragrantissima may establish additional relationships with fungi in wild conditions.