The rapid loss of native orchid habitat throughout ecologically important areas (e.g., Florida) has prompted researchers to develop appropriate plans for the propagation and reintroduction of many native orchid species. Ideally, symbiotic orchid seed germination methods are utilized in the production of orchid seedlings to be used in plant reintroduction programs. In the current study we (1) describe an efficient symbiotic seed germination protocol to germinate seeds of the rare sub-tropical terrestrial orchid Habenaria macroceratitis; (2) discuss the in vitro fungal specificity demonstrated by this species; and (3) describe the effects of three photoperiods (0/24 h, 16/8 h, 24/0 h L/D) on in vitro symbiotic seed germination of H. macroceratitis. Six fungal mycobionts were isolated from both vegetative and flowering plants of H. macroceratitis from two geographically distinct sites. Symbiotic seed germination percent was highest (65.7%) and protocorm development was most advanced (Stage 2) when seeds were cultured with fungal mycobiont Hmac-310. Seeds of H. macroceratitis demonstrated a degree of specificity toward fungal mycobionts isolated from plants originating from the same site where seed was collected. Continual darkness (0/24 h L/D) inhibited initial seed germination (Stage 1; 17.1%), but stimulated subsequent protocorm development (Stage 2; 53.5%). These findings will aid in developing an efficient symbiotic seed germination protocol for the conservation of this rare Florida terrestrial orchid, and may prove useful in the conservation of other sub-tropical terrestrial orchid species.