Rising rates of mental health problems and nonsuicidal self-injury (NSSI) among adolescents highlight the need for preventive interventions and the lack of evidence regarding such measures. To date, few studies have investigated school-based prevention programs targeting NSSI. In this study, a whole-school preventive intervention was carried out at Swedish lower secondary schools and evaluated in a cluster-randomized controlled trial. The whole-school intervention included classroom-based modules focusing on mental health and NSSI directed at students, psychoeducational webinars on NSSI directed at parents and teachers, and a two-day workshop on NSSI and suicidality for school health staff. Data were collected from students (N = 183, age M = 14.17, SD = 0.55, 58% female) at baseline and three- and six-month post-intervention follow-ups. Analyses revealed a significant decrease in three-month NSSI frequency and a significant difference in mental health-related stigma awareness in the intervention group compared to controls. Regarding other outcomes (NSSI onset, attitudes toward help-seeking, perceived social support, health-related quality of life, emotion regulation and self-criticism), no significant effects were found. Effects moderated by gender and history of NSSI were found, underscoring that the outcomes of universal prevention are not always uniformly distributed. Main conclusions were that whole-school prevention can be effective in reducing NSSI frequency as well as affecting the awareness of mental health-related stigma.