HIV disproportionately affects key populations (MSM, transgender people, sex workers and psychoactive substance users), who face greater social vulnerability and limited healthcare access. This study aimed at analyzing sexual orientation and gender identity and their association with sexual practices, sexually transmitted infections and psychoactive substance use among PrEP users. A cross-sectional study was conducted between January 2018 and June 2021 with 736 Brazilian PrEP users from a health service in São Paulo. Sociodemographic data, sexual behaviors, STI history (past 3 months) and psychoactive substances use (past 3 months) were extracted from clinical records. The associations were analyzed using binomial logistic regression ( < 0.05). Most of the participants were cisgender men (93.4%) and homosexual (84.8%), with a mean age of 34.9 years old. Condomless sex was reported by 98.5%, and 18.4% had some recent sexually transmitted infection, mainly syphilis. Psychoactive substance use was reported by 55.4%, especially marijuana, club drugs, erectile stimulants and poppers. Transgender and cisgender women were more likely to report sex work and crack use. Homosexual and bisexual participants had higher odds of using erectile stimulants. The study reveals key links between gender, sexual orientation and risk behaviors, highlighting the need for inclusive, targeted prevention.