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Rationale: To examine the acute effects of ayahuasca use and their relationship to sub-acute changes in affect and mindfulness in a non-clinical sample, addressing the need for a better understanding of ayahuasca's immediate and short-term impacts as interest in its use grows., Objectives: Using prospective ecological assessment, this study investigates how ayahuasca used at a 4-day retreat affects positive/negative affect and mindfulness skills in daily living compared to pre-retreat. Additionally, we explore acute psychedelic experiences during the ayahuasca retreat, assessed retrospectively 1-2 days post-retreat, as potential mechanisms for theorized effects in daily living post-retreat., Methods: Thirty-six participants reported positive/negative affect and mindfulness skills three times daily for 5 days before and after the retreat. Baseline assessments included lifetime psychedelic experience, and post-retreat assessments covered acute ayahuasca experiences. Mixed-effect linear models were used to analyze the data., Results: Post-retreat, we observed reduced negative affect, increased positive affect, and enhanced mindfulness skills in daily living. Ayahuasca-induced acute experiences, such as time/space transcendence, emotional breakthrough and challenging experiences predicted greater subacute positive affect. Notably, none of these experiences were linked to subacute improvements in negative affect or mindfulness. No participants showed clinically significant adverse responses post-retreat, and only 5.5% exhibited some degree of potentially clinically significant deterioration in affect., Conclusions: Ayahuasca use may lead to improvement in mood and mindfulness skills, and key acute psychedelic experiences induced by ayahuasca may be important to some of these salutary effects, positive affect in particular., Competing Interests: Declarations. Ethical approval: The study received human subjects research ethics approval from the institutional review board of the Faculty of Social Welfare & Health Sciences, University of Haifa [#214/22]. All participants gave their written informed consent. Conflict of interest: The authors have no relevant financial or non-financial interests to disclose., (© 2024. The Author(s).)