Introduction Alcohol use and drug use are common behaviours among young people. STI positivity is higher in young people than in people aged above 25 years. While there is an increasing amount of knowledge about drug use during sex among men who have sex with men (MSM), data on this behaviour among young women and heterosexual men are scarce. Therefore, this study aims to assess the proportion and characteristics of women and heterosexual men aged under 25 years reporting alcohol and/or drug use during sex and its association with STI positivity. Methods Surveillance data of heterosexual individuals younger than 25 years visiting two Dutch STI clinics between 2016 and 2019 were assessed (n=11 714). We used multivariable logistic regression analyses to assess associations between alcohol and drug use during sex and STI positivity (Chlamydia trachomatis and/or Neisseria gonorrhoeae diagnosis), adjusting for sociodemographic characteristics (sex, age, ethnicity, educational level, socioeconomic status and urbanisation) and sexual behaviour (condom use, number of sex partners). Results Alcohol use during sex was reported by 45.3% (5311/11 714; 49.5% in men vs 43.2% in women, p<0.001) and drug use during sex by 22.0% (2580/11 714; 30.7% in men vs 17.6% in women, p<0.001). The most reported drugs were cannabis (17.9%), ecstasy (XTC)/methylenedioxymethamphetamine (MDMA) (6.9%) and cocaine (4.7%). The use of at least one of the following drugs (XTC/MDMA, cocaine, speed, ketamine, gamma-hydroxybutyrate (GHB)/gamma-butyrolactone (GBL), heroin, crystal meth and/or designer drugs) was significantly associated with STI positivity after adjustment for sociodemographic characteristics (adjusted OR (aOR): 1.3, 95% CI 1.1 to 1.4), but this association did not remain significant after adjustment for sexual behaviour (aOR: 1.12, 95% CI 0.94 to 1.34). Significant associations between drug use during sex and inconsistent condom (aOR: 2.5, 95% CI 1.9 to 3.2) use and having four or more sex partners (aOR: 3.2, 95% CI 2.8 to 3.6) ...