The orchid genus Vanilla, subfamily Vanilloideae, is distributed globally throughout the tropics with over 100 species. Six species inhabit southern Florida and only four are considered native: Vanilla barbellata, V. dilloniana, V. mexicana, and V. phaeantha. Vanilla dilloniana and V. mexicana are already considered locally extinct in Florida, while V. barbellata and V. phaeantha's distribution is limited to protected areas. All four of these species are considered rare, threatened, and endangered in Florida. Their habitats face numerous environmental threats including loss of pollinators, seed dispersers, and habitat fragmentation, as well as anthropogenically driven threats such as habitat degradation and the effects of climate change like sea-level rise. Considering these escalating threats conservation efforts are needed now more than ever. Yet, little is known about their genetic structure, mycobionts, and mycobiont compositions. Through genotyping-by-sequencing (GBS) we determined genetic diversity, evidence of gene flow, and low heterozygosity of disjunct populations of sampled Vanilla barbellata populations. Additionally, we isolate and identify culturable endophytic fungi to ascertain their diversity and to acquire fungi with the potential to germinate orchid seeds. We were able to isolate and identify a wide variety of culturable fungi, from latent pathogen and non-mycorrhizal fungi to dark septate endophytes. Furthermore, with next-generation sequencing we explore and identify the composition and diversity of the mycobiomes of Vanilla barbellata and V. phaeantha and their different compartments. Counter to part of our initial hypotheses, we did not detect substantial differences of the mycobiome, between the two species, yet as we anticipated significant differences are evident between plant compartments. Data acquired in this study provide a foundational baseline for Vanilla conservation efforts that integrate their ecology, with a focus on the declining habitats of southern Florida. Accordingly, future conservation efforts that consider the relocation of these Vanilla species via assisted migration or ex situ cultivation should consider the fungal community compositions found here. Adamantly and with the support of these findings, we recommend immediate conservation efforts focused on symbiotic seed germination and assisted migration of these imperiled species to habitats beyond reach of the threats of climate change.