Indigenous Amazonian shamanic ayahuasca practice is embedded in a nature-based context and is employed as an ecological mediating agent and in collective environmental decision-making processes by some of the groups that use it. Phenomenologically, the ayahuasca experience is often rich in nature-based themes and content, and its usage has been associated with eliciting shifts in perspectives and attitudes towards nature. In this proof-of-concept study, participation in an ayahuasca retreat in a traditional Indigenous Amazonian context (with a mean of 6.31 ceremonies attended) was associated with significant increases in nature relatedness directly following ayahuasca retreats as assessed via the Short form Nature Relatedness Scale (NR-6) and The Extended Inclusion of Nature in Self scale (EINS), and improvements in depression and stress as assessed via the Depression, Anxiety and Stress Scale (DASS-21) were also found, but not on the anxiety subscale. Furthermore, a significant negative correlation with moderate effect size was found between changes in nature-relatedness and stress, suggesting that an increase in nature relatedness is associated with decreased stress levels after attending Amazonian ayahuasca retreats in our sample. It is currently unclear if the changes seen were due to consumption of the ayahuasca brew, participation in ceremony, or the retreat setting itself. Although this pilot study suggests a potential therapeutic role for Amazonian ayahuasca retreats as a multidimensional intervention, further work is required to assess the role of possible mediators underlying such shifts, while evaluating to what extent these are sustained for long term.