Nitrogen (N) pollution is increasingly recognized as a threat to biodiversity. However, our understanding of how N is affecting vulnerable species across taxa and broad spatial scales is limited. We surveyed approximately 1400 species in the continental United States listed as candidate, threatened, or endangered under the US Endangered Species Act (ESA) to assess the extent of recognized N-pollution effects on biodiversity in both terrestrial and aquatic ecosystems. We found 78 federally listed species recognized as affected by N pollution. To illustrate the complexity of tracing N impacts on listed species, we describe an interdisciplinary case study that addressed the threat of N pollution to California Bay Area serpentine grasslands. We demonstrate that N pollution has affected threatened species via multiple pathways and argue that existing legal and policy regulations can be applied to address the biodiversity consequences of N pollution in conjunction with scientific evidence tracing N impact pathways.