Since 1998, Lyme disease cases have increased in south-central Pennsylvania, which includes Gettysburg National Military Park (NMP). Limited information is available about tick populations or pathogens in this area, and no data regarding frequency of tick bites or prevention measures among Gettysburg NMP employees are available. To address these gaps, ticks were collected, classified, and replaced (to minimize disruptions to tick populations) at two sites within Gettysburg NMP during April-September, 2009, among eight nonremoval samplings. On two additional occasions during May and June, 2009, ticks were collected and removed from the two original sites plus 10 additional sites and tested for tick-borne pathogens by using PCR. A self-administered anonymous survey of Gettysburg NMP employees was conducted to determine knowledge, attitudes, and practices regarding tick-borne diseases. Peak Ixodes scapularis nymph populations were observed during May-July. Of 115 I. scapularis ticks tested, 21% were infected with Borrelia burgdorferi, including 18% of 74 nymphs and 27% of 41 adults; no other pathogen was identified. The entomologic risk index was calculated at 1.3 infected nymphs/hour. An adult and nymph Amblyomma americanum were also found, representing the first confirmed field collection of this tick in Pennsylvania, but no pathogens were detected. The survey revealed that most park employees believed Lyme disease was a problem at Gettysburg NMP and that they frequently found ticks on their skin and clothing. However, use of personal preventive measures was inconsistent, and 6% of respondents reported contracting Lyme disease while employed at Gettysburg NMP. These findings indicate a need to improve surveillance for tick bites among employees and enhance prevention programs for park staff and visitors.