Bhutan continues to report sporadic cases of various forms of leishmaniasis and has yet to receive elimination certification for visceral leishmaniasis. In the present study, the knowledge, attitudes, and practices regarding leishmaniasis among healthcare professionals were assessed in tertiary care hospitals in Bhutan. This was a cross-sectional study conducted among healthcare professionals at referral hospitals. The knowledge questions were scored with one point awarded for each correct response. A cumulative score of ≥70% was categorized as good knowledge. There were 343 participants in total, 59 (17.0%) of whom had managed leishmaniasis in their careers. Thirty (6.0%) knew the forms of leishmaniasis reported in Bhutan, 197 (58.2%) knew the case definition, 199 (55.6%) knew the screening test for visceral leishmaniasis, and 192 (54.3%) knew the confirmatory test. A total of 151 (38.8%) participants knew the first-line oral treatment for leishmaniasis. The median knowledge score was 6 of 20 (interquartile range: 5-8). A total of 60 (13.8%) participants had good knowledge. Participants who had read the national guidelines (adjusted odds ratio: 8.04; 95% CI: 2.95-21.86; P <0.001) had higher odds of demonstrating good knowledge. A total of 47 (13.0%) participants reported offering the rK39 test in the past 12 months. The majority demonstrated a willingness to learn and engage in health advocacy, prevention, and control activities. The majority lacked adequate knowledge on the forms of leishmaniasis, screening and diagnostic tests, drugs for management, and case follow-up. The number of screening tests offered to patients was low. The majority of participants were willing to engage in continuing medical education, advocacy, and health education sessions.