The use of mycorrhizal fungi to enhance orchid seed germination and seedling growth is a promising approach for orchid propagation and conservation, but practical applications remain limited. In this study, we developed a direct seeding technique based on mycorrhizal symbiosis using Dendrobium officinale. Seeds were inoculated with two fungal strains, Serendipita officinale (SO) and Serendipita indica (SI), individually or in combination, and cultivated on three substrates to identify optimal fungus–substrate combinations. SO achieved the highest germination rate (52.9 ± 5.6%) at 30 days on substrate 1 but declined at later stages, while SI performed best on substrate 3 at 30 days (72.3 ± 6.7%) but was less effective after 90 days. The SO and SI mixture showed strong synergistic effects on substrate 1, with peak germination (48.7 ± 5.9%) and seedling formation (45.6 ± 5.1%) at 120 days. Substrates 1 and 3 significantly outperformed 2 (p < 0.05), with 1 promoting rapid early germination and 3 favoring long-term seedling establishment. In contrast, controls without fungi showed less than 5% for all indices, confirming the necessity of symbiotic fungi. Microscopic observations revealed typical orchid mycorrhizal structures and dynamic hyphal turnover, providing histological evidence of the symbiotic mechanism. These findings establish a practical framework for mycorrhizal-assisted propagation and contribute to the ecological cultivation and conservation of D. officinale.