Mental health problems among medical students are a worrisome issue; recent studies have shown that one-third may be suffering major depressive disorder and one out of ten had suicidal ideation. Few studies have evaluated the association of medical students' mental health and their sexual orientation. This study aimed to evaluate differences in mental health indicators among medical students with diverse sexual orientations at a South American medical school. This study is a secondary analysis of cross-sectional data obtained through an electronic survey. The survey assessed demographics, academic variables, and several mental health scales and indexes, including: World Health Organization Well-being Index, Satisfaction With Life Scale, Family APGAR (Adaptability, Partnership, Growth, Affection, and Resolve), Self-Reporting Questionnaire, Athens Insomnia Scale, Eating Attitudes Test, and Alcohol Use Disorders Identification Test. Sexual orientation was assessed using self-identification and responses dichotomized as heterosexual and non-heterosexual. 554 students completed the survey (response rate: 70%). Mean age was 20.6 years, and the sample was 58.7% women. Eighty-two participants (14.8%) self-identified as non-heterosexual; this group comprised mostly males, fewer of whom lived with their family, and more of whom used loans or scholarships to pay university tuition fees. After adjustment for sex and tuition fee payment, non-heterosexual orientation was significantly associated (adjusted Odds Ratios [aOR] above 3.00) with rating mental health as bad, self-perceiving a need for mental health evaluation/treatment, and reporting last-year use of psychiatric medication. Lastly, non-heterosexual respondents reported more frequent psychiatric symptoms (depression/anxiety scores, suicidal ideation, eating disorder symptoms and substance use) with an aOR between 2.17 and 2.51. This study suggests that self-identified non-heterosexual medical students exhibit worse mental health outcomes evaluated through validated self-report scales and subjective perception of mental health status. This report specifically indicates that non-heterosexual medical students report family dysfunction more often and have less social support, which serve as additional risk factors. Future studies must assess social support, clarify the impact of family and peer support in mental health problems, and explore students' views on their sexual identity and the burden imposed by experiences of discrimination.