Tobacco, alcohol and cannabis are commonly used among university students. However, student lives and their substance use have changed dramatically since the start of the COVID-19 pandemic. This study investigated the impact of COVID-19 on (trends in) weekly smoking, weekly binge drinking and weekly cannabis use in Dutch university students and investigated associated student-, study- and COVID-19-related characteristics. Between April and June 2020, several Dutch higher educational institutes invited their students to participate in an online survey. Data of 9967 students (Mage = 22.0 (SD = 2.6); Nfemale = 7008 (70.3%)) were available for analyses. Overall, weekly smoking remained stable (±11.5%), weekly binge drinking decreased (from 27.8% to 13.9%) and weekly cannabis use increased (from 6.7% to 8.6%). Male gender, not living with parents, being a bachelor student, having less financial resources and less adherence to the COVID-19 measures were found to increase the risk of substance use (before/during the first COVID-19 lockdown). Additionally, male gender, not living with parents, being a bachelor student, not being born in the Netherlands and having a student loan contributed to the likelihood of increased substance use during COVID-19. Patterns of characteristics contributing to the likelihood of decreased weekly substance use during COVID-19 were less clear. The risk factors male gender, not living with parents and being a bachelor student do not only contribute to the likelihood of using substances but also contribute to the likelihood of increased use during a lockdown. Prevention and intervention programs should especially target these risk groups.