Introduction: Adolescents are particularly vulnerable to risky behaviors, such as the use of psychoactive substances (alcohol, tobacco, and illegal drugs), which can lead to other disorders throughout life. As substance use has risen over the years, the integration of new perspectives into the culture of prevention and care in youth and the general population is important. This study aimed to evaluate the relationships between dispositional mindfulness, spirituality and religion, and their role as protective factors of the psychoactive substance use in adolescents, analyzing additionally the differences in mindfulness and spirituality scores between substance and non-substance users, and between the different religious/spiritual groups.Method: A cross-sectional study was carried out, in which 433 adolescent high school students between the ages of 14 and 19 were evaluated (Mean = 16.27 ± .91). Statistical correlation tests were performed to assess the relationship between variables, in addition to Student t-test and one-way ANOVA to explore differences in mindfulness and spirituality scores of substance and non-substance users, and between religious/spiritual groups, respectively. Finally, by means of a binary logistic regression analysis, the degree of prediction of psychoactive substance use was evaluated.Results: The sample reported higher consumption of psychoactive substances compared to the last national survey. Dispositional mindfulness was positively correlated with spirituality and religion, and inversely correlated with substance use, with mild to moderate strength. Additionally, individuals who used psychoactive substances had significantly lower mindfulness scores than those who had never used it at all, but no differences were found regarding spirituality scores. The scores in mindfulness and spirituality were significantly higher in those who formally belonged to a religion. Finally, belonging to a religion, followed by dispositional mindfulness were the best predictor variables as a protection factor for alcohol and drug consumption, while spirituality was not a variable that significantly contributed to the models and was therefore eliminated.Discussion or Conclusion: Dispositional mindfulness, spirituality, and religion are interrelated variables. Belonging to religion and dispositional mindfulness may be a better protective factor against substance use in adolescents than spirituality. Dispositional mindfulness was also greater in those who formally belonged to a religion. Finally, the psychoactive substance use could alter mindfulness-related abilities, but more research is needed to complement and clarify these results in the Mexican population.