Eastern Bluebirds (Sialia sialis) do not remove nest materials; therefore associated microorganisms remain in the cavity and can potentially colonize subsequent nestlings. The impact of nest reuse or removal on bacterial communities was examined during the 2012 breeding season in western Pennsylvania. Nest success, number of fledglings produced, and nestling body condition were assessed; bacterial communities were profiled via terminal restriction fragment length polymorphisms (T-RFLP) in which one fragment approximates one bacterial species. The composition of bacterial communities varied between treatments; in particular, reused nests had fewer terminal restriction fragments and a lower diversity index. In contrast, mean nest success, number of fledglings, and body condition did not differ by treatment. Seclusion of nest material in nest boxes may result in a bacterial community in reused nests that is less diverse due to less variable conditions in the nest box.