Washington State's only endemic fish, Olympic mudminnow (Novumbra hubbsi), was listed as state “Sensitive” in 1999 due to a small range and concern about population declines and threats, including impacts from nonnative competitors and predators. We investigated the trophic ecology of Olympic mudminnow to identify habitat and foraging specialization across three sites in Lake Ozette (Olympic National Park, Washington), and evaluate evidence for resource interactions with sympatric fish species. Stable isotopes of carbon and nitrogen from dominant co-occurring fish—yellow perch (Percaflavescens), prickly sculpin (Cottus asper), and northern pikeminnow (Ptychocheilus oregonensis)—were evaluated for evidence of competition with and predation on Olympic mudminnow using trophic niche overlap and stable isotope mixing models. Evidence from co-occurrence and isotope analyses supported spatial and trophic segregation of Olympic mudminnow, and reduced likelihood of competitive and predatory relationships. Of the sympatric fish examined, prickly sculpin may be the most likely to experience biotic interactions with Olympic mudminnow. These results are consistent with other research demonstrating strong habitat and microhabitat specialization by Olympic mudminnow and suggest that access to these favored habitats may be an important mediating factor in defining the potential for biotic interactions with other species, including nonnative fishes.