Purpose: To investigate the homophobia level among the healthcare professionals working in primary healthcare services and the affecting factors. Design and Methods: This descriptive and cross-sectional study conducted between November 2018 and April 2019, included 184 healthcare professionals. Findings: The mean total score of the Hudson and Ricketts Homophobia Scale was 103.55 ± 30.47. There was a statistically significant difference between the marital status of the healthcare professionals, what they felt during the care/treatment of lesbian, gay, bisexual, transgender, and intersex (LGBTI) individuals, level of knowledge about such individuals, willingness to know more about them, and the status of having LGBTI acquaintances and the median homophobia score.