The spectral bat (Vampyrum spectrum), the largest bat species in the Americas, is considered Near Threatened by the International Union for Conservation of Nature and is listed as a species of special concern or endangered in several countries throughout its range. Although the species is known as carnivorous, data on basic ecology, including habitat selection and primary diet items, are limited owing to its relative rarity and difficulty in capturing the species. Leveraging advances in DNA metabarcoding and using radio-telemetry, we present novel information on the diet and movement of V. spectrum based on locations of a radio-collared individual and fecal samples collected from its communal roost (three individuals) in the Lowland Dry Forest of southern Nicaragua. Using a non-invasive approach, we explored the diet of the species with genetic markers designed to capture a range of arthropods and vertebrate targets from fecal samples. We identified 27 species of vertebrate prey which included birds, rodents, and other bat species. Our evidence suggested that V. spectrum can forage on a variety of species, from those associated with mature forests to forest edge-dwellers. Characteristics of the roost and our telemetry data underscore the importance of large trees for roosting in mature forest patches for the species. These data can inform conservation efforts for preserving both the habitat and the prey items in remnants of mature forest required by Vampyrum spectrum to survive in landscape mosaics.