Orchid–mycobiont specificity in the Orchidaceae was considered controversial and not well understood for many years. Differences in mycobiont specificity during germination in vitro vs in situ have lead some to consider orchid–mycobiont specificity as being generally low; however, others have suggested that specificity, especially in vitro, is surprisingly high. Mycobiont specificity may be genus or species specific. An in vitro symbiotic seed germination experiment was designed to examine mycobiont specificity of the endangered Florida terrestrial orchid Spiranthes brevilabris using mycobionts isolated from both the study species and the endemic congener Spiranthes floridana. In a screen of mycobionts, isolates Sflo-305 (99.5%), Sflo-306 (99.5%), and Sflo-308 (89.9%) (originating from S. floridana) supported higher initial (stage 1) seed germination than isolate Sbrev-266 (32.4%) (originating from S. brevilabris) after 3 wk culture. However, only isolate Sbrev-266 supported advanced germination and protocorm development to stage 5 (53.1%) after 12 wk culture. These findings suggest that S. brevilabris maintains a high degree of mycobiont specificity under in vitro symbiotic seed germination conditions. High orchid–mycobiont specificity in S. brevilabris may be indicative of the rare status of this orchid in Florida.