Nurses are managing huge number of patients infected with human immunodeficiency virus (HIV), which made them highly vulnerable to HIV infection through occupational exposure such as needle stick injuries and splashing of blood/bodily fluids on mucosal surface. This made the practice of post exposure prophylaxis (PEP) for HIV crucial among nurses. Therefore, our study aimed to assess knowledge, attitude and practice of PEP for HIV among nurses in Bhutan. A cross-sectional study was conducted among 221 registered nurses working at Jigme Dorji Wangchuck National Referral Hospital, Bhutan between April and June 2017. A structured self-administered questionnaire was used to collect data and analysed using SPSS version 21. Majority (80.1%) of our participants had poor knowledge regarding PEP for HIV. Although half (51.1%) of our participants had heard about PEP, only 3 (1.4%) attended a formal training on PEP for HIV. However, a significant proportion of nurses (92.3%) had positive attitude towards PEP for HIV. Out of 221 respondents, 95(43%) had been exposed to needle stick injuries and splashing of blood/bodily fluids while managing patients. Despite significant number of exposures, only 2 (2.1%) of them took PEP and completed 28 days of prophylaxis. Lack of protective barriers at work place (56.8%) and poor knowledge on personal protective equipment (14.7%) were major perceived causes of exposure among study participants. No PEP service (30.2%) and lack of support to report incidents (22.6%) were two major reasons leading to failure of PEP practice among exposed individuals. Despite positive attitude exhibited by majority of our respondents, the level of knowledge and practice of PEP for HIV among nurses was very low. Therefore, a formal training on PEP and 24 hours accessible PEP service with proper guidelines are recommended to improve the overall knowledge and practice of PEP against HIV among nurses.