Compatible fungal partners of orchids can significantly enhance seed germination and increase seedling establishment under both in vitro and in situ conditions. This study isolated 14 Tulasnella isolates from five-year-old potted plants of three D. officinale cultivars. Three phylogenetically representative strains (Dca122, Dca222, and Dca113) and two additional orchid mycorrhizal fungus (OMFs, ML01 and Pi) were selected to evaluate their effects on D. officinale seed germination and seedling development in vitro, and subsequent seedling growth under greenhouse conditions. All five OMFs supported seed germination and seedling development in vitro. Notably, Dca113, Pi, and ML01 exhibited the most pronounced effects, producing protocorms 3–4 times larger in volume than controls. By day 25, 37.54%, 37.34%, and 42.6% of protocorms developed cotyledons with these isolates, respectively. Furthermore, after 120 days, ML01 and Dca113 treatments yielded 35.6% and 30.68% autotrophic seedlings with fully differentiated roots. Under greenhouse, ML01, Pi, and Dca122 significantly enhanced fresh weight accumulation, plant height, and stem node number in potted seedlings. In contrast, Dca222 primarily stimulated sprouting tillers and adventitious root formation. Our results demonstrate that the mycorrhizal effectiveness of OMFs from different hosts varies significantly in D. officinale. ML01 and Dca113 are ideal candidates for reintroduction programs due to their strong promotion of seed germination and rapid formation of rooted seedlings. ML01 proved the most effective OMF for enhancing growth in potted seedlings, while Dca222 demonstrated potential for co-inoculation strategies.