Recent studies have recognized the importance of non-pharmacological factors such as setting to induce or promote mystical experiences or challenging experiences among ayahuasca users. This study aimed to evaluate the association between the setting in which ayahuasca is consumed and the intensity of mystical and challenging experiences considering three ayahuasca using traditions (União do Vegetal, Santo Daime and neo-shamanic groups). A cross-sectional analysis was performed on survey data collected online from 2,751 participants. The Setting Questionnaire for the Ayahuasca Experience (SQAE) was used to evaluate six dimensions of the setting characteristics. The Mystical Experience Questionnaire (MEQ) and the Challenging Experience Questionnaire (CEQ) were used to quantify the psychedelic experience. Ratings on every SQAE setting dimension were negatively correlated with ratings of the CEQ (r values between 0.21 and 0.36) for all ayahuasca using traditions. Regression analysis revealed that ratings on four SQAE dimensions (Social, Comfort, Infrastructure and Decoration) explained 41% of the variance in CEQ ratings. Associations between SQAE and MEQ ratings were relatively weak and confined to the dimensions Leadership and Comfort, explaining 14% of the variance in MEQ ratings. Ratings of Social context were higher among members of União do Vegetal compared to Santo Daime and neo-shamanic members. Ratings of Infrastructure, Comfort and Decoration were more consistently correlated with MEQ in the neoshamanic tradition compared to the other traditions. This study shows that the setting is an important moderator of a challenging experience under ayahuasca. Maximizing the quality of the setting in which ayahuasca is taken will reduce the chance of a challenging experience while contributing positively to a mystical experience. The present findings can be considered when designing rituals and the (social) environment of ayahuasca ceremonies, and indicate that the SQAE questionnaire can be employed to monitor the influence of ceremonial settings on the ayahuasca experience.