Prevalence rates of compliance with anti-COVID measures have been widely studied, but little is known about this issue in early adolescence. Moreover, the relation between substance use and compliance with anti-COVID regulations is still unexplored. Thus, this study aimed to determine the level of compliance with anti-COVID measures by adolescents and the link between substance use and compliance with anti-COVID regulations. This was a cross-sectional study including 909 participants (Mage = 12.57; SD = 0.81). The most complied measure was mask-wearing, followed by avoiding hug/kiss friends and, finally, social distancing. All substance use negatively correlated with compliance with measures. However, strong alcohol and tobacco were the only substances significantly related to less compliance of anti-COVID measures after controlling for covariates. These results provide evidence about the relation between substance use and compliance with anti-COVID measures. Strategies addressed to decrease substance use could be effective to reduce behaviours associated with coronavirus transmission.