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Objective: Psychoactive substance use is prevalent among young people in Nigeria. However, the dearth of accessible and good-quality treatment for substance use disorders in Nigeria creates a treatment gap. This study evaluated the effectiveness of a substance use prevention education programme (SUPEP) for young people in Nigeria. Design: A quasi-experimental study using a pre- post-test was conducted. The experimental group (n = 100) and comparison group (n = 100) comprised students in four senior secondary schools. Data were collected using a validated self-report questionnaire and the WHO Alcohol, Smoking, and Substance Involvement Screening Test (ASSIST) version 3.0. Paired sample t-tests, analysis of covariance (ANCOVA) and generalised and linear mixed models (GLMM) were used for data analysis. Setting: Two hundred young people aged 14-20 years prone to substance use participated in SUPEP in four secondary schools in Enugu State, Southeast, Nigeria. Results: Students' psychoactive substance knowledge and substance use attitudes in the experimental group (p < 0.001) improved significantly post-intervention. ANCOVA results showed that the mean psychoactive substance knowledge score increased significantly between the time points, F(1, 123) = 8.89, p = 0.003. In contrast, mean psychoactive substance use attitudes reduced significantly between the same time points, F(1, 123) = 12.390, p = 0.001. However, no significant difference between the groups was observed in the mean psychoactive substance use behaviour post-intervention. Conclusion: The school-based SUPEP increases psychoactive substance knowledge and improves attitudes. However, the intervention did not reduce substance use among adolescents.