The objective of the present study was to assess HIV-related knowledge, attitudes, and practice of health care workers (HCWs) in Montenegro. A cross-sectional study was conducted in the University Clinical Centre of Montenegro in Podgorica. A self-administered anonymous questionnaire was used for data collecting. Out of 526 HCWs, 422 were included in the survey and response rate was 80%. An insufficient level of knowledge on HIV transmission and the risk after exposure was observed generally, although the knowledge was better in physicians compared to other HCWs categories. A rather high proportion of HCWs showed inappropriate attitude regarding the need of HIV testing of all hospitalized patients (64.7%) and obligation of HIV+ patient to report his/her HIV status (88.9%) in order to practice universal precaution. Additionally, 6.2% HCWs would refuse to treat an HIV+ patient. More than a half (55.7%) of study participants were educated in HIV/AIDS and 15.9% of them were HIV tested. Majority of HCWs (67.5%) always applied universal precautions during their daily work with patients. In spite of applying protective devices, number of accidents was great. A continuous education is necessary to increase the level of knowledge of HCWs about the risk of infection at the workplace. This would potentially influence the modification of their attitudes regarding HIV patients and improve prevention at the workplace. Continuous research regarding the professional risk would provide better health and safety among medical staff.