Background. Little attention has been paid to the role of poor mental health among young people with regard to HIV risk behaviour and HIV prevention in Africa. Objective. To determine the association between mental health, substance use and HIV sexual risk behaviour among a sample of university students in South Africa. Methods. A cross-sectional survey was conducted among undergraduate students who were recruited conveniently from public campus venues at the University of Limpopo Medical University of Southern Africa (Medunsa) campus. The sample included 722 university students (57.6% men and 42.4% women) with a mean age of 21.7 years (standard deviation ±8.8). Results. Of the 722 students, 39.5% reported depression, 23.4% screened positive for post-traumatic stress disorder (PTSD), 22% reported hazardous or harmful alcohol use, 33% reported ≥2 sexual partners in the past 12 months, 50% reported inconsistent condom use, 46% reported unknown HIV status of a sexual partner and 20% reported alcohol use in the context of sex in the past 3 months. In multivariate analysis, HIV risk behaviour was associated with, among men, hazardous or harmful alcohol use and having screened positive for PTSD, and among women, being in the 4th or more year of study and current cannabis use. Conclusion. Poor mental health, including substance use, was found to be associated with HIV risk behaviour. Co-ordinated mental health and sexual and reproductive health services that meet the needs of university students would be desirable.