The presence of preputial Demodex species in four captive, wild-caught big brown bats (Eptesicus fuscus) in eastern Tennessee, USA, examined between 2008 and 2010 is reported. In three of four cases, there was associated folliculitis, dermatitis, or preputial adenitis. There were no clinical signs directly attributable to the mites, although penile prolapse was present in one case. Mites were retrieved from preputial skin samples of other archival big brown bats by potassium hydroxide digestion and analyzed by polymerase chain reaction (PCR). DNA sequencing of the 16S rRNA gene revealed 99.6% homology to Demodex canis, although morphologic differences distinguish the two species. Mites of this report differ in anatomic location from Demodex spp. previously reported in bats and represent a species unreported to public databases.