For effective conservation of threatened orchids, identifying root-associated fungi and assessing their activity in seed germination is important for establishing conservation protocols. Therefore, our study investigated the diversity of Spiranthes spiralis‘s root-associated fungi. According to the culture-dependent approach, 37 endophytic fungi were isolated from the roots and morphologically and molecularly identified. It was determined that the dominant fungal species in the roots was the genus Tulasnella. For the first time in Türkiye, the Thanatephorus fusisporus species was isolated from roots. The germination efficiency of the isolated fungi in the symbiotic culture of S. spiranthes seeds was evaluated. VY 25 (Tulasnella) isolate isolated in April showed the highest germination rate (73.77%). VY 4, VY 18, VY 25, (Tulasnella) isolates promoted germination and seedling development. Thanatephorus (46.79%) and Ceratobasidium (32.42%) were not effective in germinating seeds. The study revealed that the fungal partner varied according to developmental stages and months. This study contains the first molecular data for organisms isolated from roots in Türkiye. According to these results, fungi that promote seed germination and plant growth can be recommended for the conservation and reintroduction of endangered temperate orchids.