Ex situ conservation, an important way to increase the survival and sustainability of endangered species, is widely used in the conservation of endangered orchids. However, long-term ex situ conservation might affect the dominant group of orchid symbiotic fungi, which are crucial for orchid growth and reintroduction. This study investigated the culturableTulasnellaspp. associated withPaphiopedilumorchids after long-term greenhouse cultivation, and identified germination-enhancing isolates. A total of 44Tulasnellaisolates were obtained from the roots of 14Paphiopedilumspp., and 29 of them were selected for phylogenetic analysis. They clustered mainly withTulasnella deliquescens,Tulasnella calospora,Tulasnella bifrons,andTulasnella irregularis, but included two potential new groups. Compared with published uncultured data, most of the isolates were grouped together with the reported types, and the dominantTulasnellaassociated withP. armeniacumandP. micranthumcould still be isolated after ten years of cultivation, most of which were the first isolation. In vitro symbiotic germination showed that certain root isolates could promote seed germination (e.g., parm152 isolated fromP. armeniacum, Php12 fromP. hirsutissimum, and prhi68 fromP. rhizomatosum). These data indicated that the dominantTulasnellatypes colonizing the roots of cultivatedPaphiopedilumare stable over time, and germination-enhancing fungi colonizing the roots would benefit for seed reproduction after population reintroduction into the wild.