U.S. National Park Service employees may have prolonged exposure to wildlife and arthropods, placing them at increased risk of infection with endemic zoonoses. To evaluate possible zoonotic risks present at both Great Smoky Mountains (GRSM) and Rocky Mountain (ROMO) National Parks, we assessed park employees for baseline seroprevalence to specific zoonotic pathogens, followed by evaluation of incident infections over a 1-year study period. Park personnel showed evidence of prior infection with a variety of zoonotic agents, including California serogroup bunyaviruses (31.9%), Bartonella henselae (26.7%), spotted fever group rickettsiae (22.2%), Toxoplasma gondii (11.1%), Anaplasma phagocytophilum (8.1%), Brucella spp. (8.9%), flaviviruses (2.2%), and Bacillus anthracis (1.5%). Over a 1-year study period, we detected incident infections with leptospirosis (5.7%), B. henselae (5.7%), spotted fever group rickettsiae (1.5%), T. gondii (1.5%), B. anthracis (1.5%), and La Crosse virus (1.5%) in staff members at GRSM, and with spotted fever group rickettsiae (8.5%) and B. henselae (4.3%) in staff at ROMO. The risk of any incident infection was greater for employees who worked as resource managers (OR 7.4; 95% CI 1.4,37.5; p=0.02), and as law enforcement rangers/rescue crew (OR 6.5; 95% CI 1.1,36.5; p=0.03), relative to those who worked primarily in administration or management. The results of this study increase our understanding of the pathogens circulating within both parks, and can be used to inform the development of effective guidelines and interventions to increase visitor and staff awareness and help prevent exposure to zoonotic agents.