Tree cavities are rarely incorporated into surveys of forest ecosystem biodiversity, due to difficultly in their systematic sampling. We examined the feasibility of using southern flying squirrel (Glaucomys volans Thomas) nest boxes for monitoring arboreal vertebrates at 11 sites within the Savannah River Site, SC. We recorded 3130 vertebrates of 11 species (3 mammals, 3 birds, 5 reptiles) using nest boxes for nesting, roosting, and foraging. G. volans represented the majority of these with 3019 individuals, but flying squirrel occupancy did not affect occupancy of boxes by other species. Upland hardwood forests had the most species that used boxes; however, due to uneven sampling, nest boxes placed in dense-canopy plantations detected the most species per box. We conclude that nest boxes are a useful means of surveying for cavity-dwelling species. We recommend a protocol that uses different size nest boxes at varying heights to accurately survey a traditionally under-sampled component of forest ecosystems, those species using tree cavities.