Background The use of psychoactive substances among adolescents is a major social and public health concern. Aim To analyze association of substance abuse and multiple drug use with health-related quality of life (HRQOL) in adolescents attending a high school in Valparaiso, Chile. Material and Methods Analytical cross-sectional study conducted in a sample of adolescents attending high school. HRQOL was assessed using KIDSCREEN-52 questionnaire and substance use was measured using the Global school-based student health survey. Participants had to complete online, self-administered, anonymous questionnaires. Multiple logistic regression analyses were conducted to calculate Odd ratios. Results A total of 550 adolescents aged 16 ± 1 years old completed the questionnaires. Thirty nine percent consumed alcohol during the last month, 31% smoked, 33% used marijuana and 33% admitted the use of multiple drugs. High-risk alcohol consumption was associated with a lower perception of psychological well-being, self-perception and school environment. This last dimension was affected in those who admitted marijuana use during the last month. Multiple drug use (three substances) was associated with a lower perception of physical and psychological well-being, self-perception, relationship with parents, family life and school environment. Conclusions High-risk alcohol consumption and multiple drug use (three substances) have a negative impact on the HRQOL of school age adolescents.