Molecular techniques allow amplification of a mitochondrial barcoding gene, cytochrome c oxidase (COI), of prey DNA in bat fecal material that can be used to identify insect species. Most studies designed to use a molecular approach for dietary studies of bats suggest that fecal material should be collected within one week of deposit to prevent environmental degradation or contamination. However, Ozark big-eared bats (Corynorhinus townsendii ingens) are highly susceptible to human disturbance. The purpose of this study was to performing a molecular dietary analysis of Ozark big-eared bats. Our study detected 40 species representing two orders (Diptera and Lepidoptera) and 11 families of insects and thus, providing new information regarding dietary habits of Ozark big-eared bats.