The fauna of water-filled tree holes in Neotropical forests is not well documented. Cumulatively, 54 macroinvertebrate and 5 vertebrate taxa were found in artificial and natural tree holes censused over four wet seasons on Barro Colorado Island, Panama. Most of the species were in the insect order Diptera, occurred as aquatic larvae in tree holes, and were detritivore/omnivores. Half (49%) of the collected species are considered specialists in this and similar container habitats, and three invertebrate taxa were previously unknown from tree holes. Successional patterns were weak in tree holes, but some taxa predictably colonized holes shortly after they were filled. The mosquito Culex urichii was more common and abundant in artificial than in natural tree holes; occurrence frequencies and densities of most other taxa were similar between hole types.