The National Wildlife Refuge (NWR) System protects ~150 million acres of land and water in the United States and provides habitat for >2,000 native vertebrates species. Although legally protected, wildlife populations within these refuges can be threatened by anthropogenic activities. The lack of knowledge about such threats has the potential to undermine biodiversity conservation. We investigated patterns of wildlife mortality among three NWRs in the northern U.S. Gulf Coast. Visual surveys (958 total search-hours) were conducted for live or dead amphibians and reptiles at Big Branch, Bogue Chitto, and Bayou Sauvage NWRs. An exceptionally high incidence of snake mortality (>800% above background, P < 0.001) was observed at one site (Haul Road, Bayou Sauvage NWR). Mortality affected seven species and recurred for 29 months. Intact carcasses were subject to histopathological examination and diagnostic testing. Cause of death was undetermined (n = 9) or attributed to various pathogens (n = 5). To investigate possible underlying causes of this unusual mortality, water exposure challenges and soil toxicological analyses (n = 6 and 11 sites, respectively) were conducted on samples from Bayou Sauvage NWR, given the known potential for anthropogenic contamination in the refuge. In a controlled experiment, survival of Anaxyrus fowlerii embryos exposed to water from Recovery Road (i.e., 250m from a landfill) and Haul Road was reduced 92% and 48%, respectively, compared to four reference sites in the refuge. Sediment analyses suggested at least seven distinct sources of contaminants in Bayou Sauvage NWR. Potential sources included a landfill, illegal dumping, vehicle emissions, pesticide migration, and an active hydrocarbon pipeline leak discovered 600 m from Haul Road. Synthesis and applications: Although further research is needed to determine whether the observed mortality and contamination are related, these collective findings suggest that anthropogenic factors may threaten the ecological integrity of one of the United States largest urban wildlife refuges. More broadly, our study highlights the critical need for species inventories, baseline data, and systematic monitoring in NWRs. Such information is essential to detecting and mitigating anthropogenic threats to biodiversity conservation in protected areas.