Abstract Background This study employs a cluster randomized controlled trial to investigate the effectiveness of a universal mindfulness-based intervention (MBI) in classrooms in improving emotional well-being and emotion regulation among adolescents with elevated ADHD symptoms. Methods The study comprised 342 students: 164 in an MBI group, 128 in a relaxation control group, and 47 in an inactive control group. Positive and negative emotional states and the emotion regulation strategies of rumination, acceptance, catastrophizing, positive reappraisal, and self-kindness were measured at the pre- (T0) and post-intervention (T9) and the 26-week follow-up (T26). Linear mixed models were used to analyze the intervention effects among all participants and separately by gender and ADHD symptom type. Results The results showed that compared to the controls, there was a beneficial effect on the MBI group concerning acceptance at T9; acceptance remained at the baseline level in the MBI group but decreased in the inactive control group. In contrast, in the inactive control group, rumination decreased at T9, and positive reappraisal increased at T26 compared to the MBI group. The MBI had no detectable effects on self-kindness, catastrophizing, or positive/negative emotional states. Findings on the emotion regulation strategies varied by gender. In boys, the MBI increased acceptance at T9 compared to the inactive control group, whereas in girls, the programme reduced catastrophizing at T9 and T26 compared to the active control group. Contrary in boys, MBI group exhibited less reduction in catastrophizing at T9 compared to the inactive control group. Conclusions Universal MBIs may have some beneficial effects on emotion regulation in adolescents with elevated ADHD symptoms. Both girls and boys might be responsive to universal MBIs as part of the school curriculum, however not all consequences are positive (especially in boys). Key messages • Universal MBIs in classrooms may have some beneficial effects on emotion regulation among adolescent with elevated ADHD symptoms; however, the results are contradictory and more research is needed. • Even though, universal MBIs may have some benefits, they may require modifications to lead to more beneficial effects on emotional well-being when used with this subgroup of students.