In 2007, a National Park Service (NPS) biologist died from pneumonic plague after unprotected exposure to an infected mountain lion. This incident increased awareness of occupational zoonotic disease transmission and prompted an assessment of employees who handle wildlife. During April–June 2009, we conducted a national online survey of NPS biologists and other wildlife workers to assess in the preceding 12 mo: 1) potential work-related zoonotic disease exposures; 2) protective practices, including use of personal protective equipment (PPE); and 3) barriers and facilitators to PPE use. Summary protective measure scores were calculated and compared with sociodemographic and work-related factors. Surveys were completed by 238 employees from 131 parks in all NPS regions. Seventy-one percent were biologists or technicians, 16% natural resource specialists or managers, and 13% had other job titles. Among a majority of respondents, interactions with animals were infrequent and occurred approximately several times per year as follows: handling live (39%), sick (43%), or dead animals (46%), and drawing blood from animals (42%). The most frequently reported protective measures used were hand hygiene and gloves. Commonly agreed-upon measures that would facilitate PPE use included having PPE stocked and readily available (92%) and having specific PPE kits for use during necropsies (91%) and in remote field settings (91%). Significantly higher summary protective measure scores were found if respondents had either read or reviewed ‘‘NPS safe work practices for employees handling wildlife’’ with their supervisor, had zoonotic disease safety or PPE use included in their employee performance appraisal plans, or had conducted a job-hazard analysis for handling wildlife. Ninety (38%) respondents reported receiving zoonotic disease training. Our findings support the development and implementation of workplace interventions to increase zoonotic disease awareness and promote a culture of prevention among wildlife professionals.