<p>Orchids normally produce large numbers of dust-like seeds that rely heavily on orchid mycorrhizal fungi (OMFs) for germination and subsequent seedling development. Using OMFs to facilitate orchid proliferation is considered an effective way for orchid conservation, but this requires obtaining optimal sources of fungi, and using fungi appropriately in conservation practices based on understanding the symbiosis between orchids and fungi in nature. In this study, we obtained four fungal isolates belonging to a <i>Tulasnella</i> species (QYM-1-QYM-4) from symbiotically formed protocorms of <i>Dendrobium huoshanense</i> by using <i>ex situ</i> seed baiting, and compared their efficiencies with other sources of OMFs on seed germination and seedling development of <i>D. huoshanense</i> in vitro and in the natural habitat. The four isolates and other two <i>Serendipita</i> fungi originally obtained from other orchid species and soil could promote seeds germinated up to seedlings with different effectiveness in vitro. The isolates QYM-1 and <i>Serendipita indica</i> SI showed high abilities to promote seedling development, and the percentages of seedlings in QYM-1 and SI treatments were 53.53 ± 3.2% and 53.65 ± 2.2% (<i>P =</i> 0.96) at 90 days after incubation, which were significantly higher than other fungal treatments. However, in natural habitat after 90 days, only 1.33 ± 1.3% seedlings developed in SI treatment while 29.67 ± 4.1% seedlings survived in QYM-1 treatment (<i>P</i> &lt; 0.05). Our results revealed that a wide range of fungi could promote seed germination up to seedling in vitro but strong ecological specificity occurred during seedling establishment in the natural habitat of in <i>D. huoshanense</i>. Therefore, we suggest isolating ecologically specific fungi from protocorms/seedlings via seed baiting, and use the obtained fungi to facilitate symbiotic seed germination for orchid reintroduction in the recipient habitats.</p>