Abstract: To found the interaction effects of orchid mycorrhiza on the growth of orchid seedlings, and screen the combination of synergistic interaction modes, 3 mycorrhizal fungi(PF02, PF06 and PF07) isolated from fresh nutritious roots of Paphiopedilum hirsutissimum were studied. Seven inoculations including single and mixed inoculation of 2 and 3 fungi were adopted, the dominant strains PF02 and PF07 were screened out by fungus-seedling symbiotic culture. The dominant strains PF02 and PF07 were screened out by fungus-seedling symbiotic culture. The relationship between inoculation method and inoculation effect in vitro was revealed, and the effects of mycorrhizal fungi interaction on the growth and physiology of P. hirsutissimum were analyzed. The result proves that mycorrhizal fungi interaction was good for biomass accumulation, protective enzyme activity and total chlorophyll content of P. hirsutissimum seedlings, such as inoculation modes with 2 mycorrhizal fungi PF02−PF06, PF02−PF07 and PF06−PF07, especially PF02−PF06, which were effective for the activities of POD, SOD, CAT and total chlorophyll increased significantly. Selecting the optimal inoculation method and the best combination of inoculation methods that could be great significance to P. hirsutissimum production.