Elucidating the diet of Neotropical migratory birds is essential to our understanding of their ecology and to their long-term conservation. Reductions in prey availability negatively impact Neotropical migrants by affecting their survival as both nestlings and adults. Beyond broad taxonomic or morphological categories, however, the diet of Neotropical migrants is poorly documented. Using the molecular techniques of DNA barcoding and next-generation sequencing, we elucidated the diet of Louisiana Waterthrush (Parkesia motacilla) nestlings in Arkansas and Pennsylvania, USA. Waterthrush have been shown to respond negatively to the reduced availability of aquatic insects in the orders Ephemeroptera, Plecoptera, and Trichoptera (EPT taxa). We hypothesized that Louisiana Waterthrush nestling diet would be primarily composed of these pollution-sensitive aquatic taxa, and that changes in the riparian insect community would be reflected in their diet. Unexpectedly, the orders Lepidoptera (92%) and Diptera (70%) occurred frequently in the diet of Louisiana Waterthrush nestlings. Among EPT taxa, only the order Ephemeroptera (61%) was frequently detected whereas Plecoptera (7%) and Trichoptera (1%) were poorly represented. The frequency at which aquatic Ephemeroptera and terrestrial Lepidoptera were detected in waterthrush nestling diet differed significantly over the nesting period in Pennsylvania but not in Arkansas, suggesting that phenological shifts in the availability of non-EPT prey taxa may be an important yet undescribed factor influencing the foraging ecology of waterthrush on the breeding grounds. Furthermore, these findings suggest that terrestrial insects may be more important to waterthrush nestlings than previously thought, which enhances our understanding of this biological indicator and Neotropical migrant.