Background: The attitudes of medical professionals towards homosexuals can influence their willingness to provide these individuals with medical help. The study evaluated the medical professionals' knowledge about homosexuality and their attitudes towards it. Subjects and methods: The sample consisted of 177 participants (physicians n=79 and students n=98). The study respondents anonymously completed three questionnaires (socio-demographic questionnaire, the questionnaire on knowledge, and the questionnaire on attitudes towards homosexuals). Results: Male and religious participants showed a lower level of knowledge and a greater tendency to stigmatize. Furthermore, the subjects who knew more about homosexuality tended to hold less stigmatizing attitude. Age group, specialty (psychiatry, gynecology, internal medicine and surgery), and student's/physician's status had no effect on stigmatization. The study showed that the final year students/ residents had more knowledge than the second year students/specialists did. Knowledge had significant negative predictive effect on attitudes in the analyzed predictive model. Conclusions: To our knowledge, this has been the first study in Serbia and Eastern Europe, which provides information on knowledge and attitudes of health professionals towards homosexuality. We would like to point out the degree of knowledge on homosexuality as a possible, but not exclusive tool in shaping the attitudes towards homosexuals and reducing stigmatization. However, regardless of the personal attitude, knowledge and variable acceptance of the homosexuals' rights, medical professionals' main task is to resist discriminative behavior and provide professional medical help to both homosexual and heterosexual patients.