Objectives Workplace health and safety is vital in every organization particularly in the healthcare settings. The aim was to assess the levels of knowledge, attitude, and practices of the healthcare professionals towards safety at Gondar University referral hospital. An institution based cross-sectional study was conducted from February to June 2018. Proportional random sampling technique was used to include 282 study participants and data were collected using a structured self-administered questionnaire and analyzed using SPSS version 20. Results Among 282 study participants, 230 (81.6%), 181 (64.2%), and 162 (57.4%) had adequate knowledge, favorable attitude, and adequate practice scores, respectively. More than half (55.3%) of the study participants were untrained. There was a high (26.6%) prevalence of needlestick injury; however, the use of post-exposure prophylaxis after potential exposures was very limited. Generally, the levels of knowledge, attitude, and practice scores among the study participants were low. Therefore, there should be adequate and consistent supply of personal protective devices and other materials used for infection prevention and control. In addition, there should be awareness raising mechanism, including the provision of job aids and periodic training. Further, comprehensive studies should be conducted by including different types and levels of health facilities.

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