Relationships among cavity-nesting birds, trees, and wood decay fungi pose interesting management challenges and research questions in many systems. Ornithologists need to understand the relationships between cavitynesting birds and fungi in order to understand the habitat requirements of these birds. Typically, researchers rely on fruiting body surveys to identify the fungal players in these relationships. Fruiting body surveys enable nondestructive sampling, but vastly underestimate fungal presence and diversity and may miss species of critical importance to cavity- nesting birds; thus new methods for such analyses are necessary. Here we present a novel technique to nondestructively sample the wood inside tree cavities, which produces samples that can be processed using DNA-based methods to identify fungi. We tested our method on Red-cockaded Woodpecker Picoides borealis excavations, half of which were from trees with Porodaedalea pini fruiting bodies. Using our new approach, we detected P. pini in 90% of the excavations in trees with fruiting bodies, but also in 60% of the excavations in trees without fruiting bodies and identified nine additional taxa of wood decay fungi that did not have fruiting bodies present. Our approach offers improved detection of fungi through non-destructive sampling of excavated cavities and we developed an improved primer specific to the fungal phylum that contains most wood decay fungi (Basidiomycota), thus providing managers and researchers a critical tool to better determine which fungi are important to cavity-nesting birds.