Healthcare Professionals Working in Schools (HPWS) are responsible for providing health services to students and play a role in providing education to prevent illnesses, including tickborne diseases (TBD). Providing TBD education to children has been shown to increase prevention behaviors and knowledge of TBD symptoms, but little is known regarding the current state of TBD awareness among HPWS. In spring 2019 we conducted a cross-sectional knowledge, attitudes, and practices (KAP) survey of HPWS in two states with a high incidence of Lyme disease (LD) to inform design of TBD prevention programs. The survey queried general knowledge of TBDs, school practices regarding TBDs, and availability of TBD resources. Overall, higher confidence, experience, risk perception, prior training on TBD, and more years employed as a HPWS were independently associated with knowledge of LD transmission, symptoms, and correct tick removal practices. State and local health departments should consider prioritizing engagement with HPWS to provide educational opportunities about tickborne diseases.