In show caves, artificial lighting is intended to illuminate striking cave formations for visitors. However, artificial lighting also promotes the growth of novel and diverse biofilm communities, termed lampenflora, that obtain their energy from these artificial light sources. Lampenflora, which generally consist of cyanobacteria, algae, diatoms, and bryophytes, discolor formations and introduce novel ecological interactions in cave ecosystems. The source of lampenflora community members and patterns of diversity have generally been understudied mainly due to technological limitations. In this study, we investigate whether members of lampenflora communities in an iconic show cave—Lehman Caves—in Great Basin National Park (GRBA) in the western United States also occur in nearby unlit and rarely visited caves. Using a high-throughput environmental DNA metabarcoding approach targeting three loci—the ITS2 (fungi), a fragment of the 16S (bacteria), and a fragment of 23S (photosynthetic bacteria and eukaryotes)—we characterized diversity of lampenflora communities occurring near artificial light sources in Lehman Caves and rock surfaces near the entrances of seven nearby “wild” caves. Most caves supported diverse and distinct microbial-dominated communities, with little overlap in community members among caves. The lampenflora communities in the show cave were distinct, and generally less diverse, from those occurring in nearby unlit caves. Our results suggest an unidentified source for a significant proportion of lampenflora community members in Lehman Caves, with the majority of community members not found in nearby wild caves. Whether the unique members of the lampenflora communities in Lehman Caves are related to distinct abiotic conditions, increased human visitation, or other factors remains unknown. These results provide a valuable framework for future research exploring lampenflora community assemblies in show caves, in addition to a broad perspective into the range of microbial and lampenflora community members in GRBA. By more fully characterizing these communities, we can better monitor the establishment of lampenflora and design effective strategies for their management and removal.