Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is a neglected parasitic infection in the United States (US). In the Southwestern US, National Park Service (NPS) employees are a unique population with potential exposure to CD. This population lives in close contact with several species of sylvatic triatomine bugs, the vectors of T. cruzi, that may enter residential buildings at night. Despite the higher potential risk of CD transmission for southwestern NPS employees, the socio-cultural factors that impact autochthonous CD transmission in the US remain unknown. To address this gap, we investigated how NPS employee knowledge and attitudes impact their triatomine preventive behaviors. We distributed a 42-item online questionnaire to NPS employees at four national parks in Arizona and Texas. We detected high self-reported bite exposure in NPS housing, despite moderate- to high-frequency of prevention behaviors. Specific behaviors, such as often or always repairing window screens, were associated with a decreased risk of putative triatomine bug exposure. Additionally, NPS employees had low knowledge of CD. For those with greater knowledge of CD, it was not associated with increased frequency of prevention behavior. We found that increased CD anxiety was associated with increased personal agency to reduce the risk of CD. These results demonstrate the influence of knowledge and attitudes regarding CD on triatomine prevention behavior within a potential high-risk population in the US, and the importance of utilizing strategies beyond provision of education to influence behaviors.