Background: Ayahuasca, a South American psychotropic plant tea containing the psychedelic 5-HT2A receptor agonist N,N-dimethyltryptamine, has been shown to increase region- al cerebral blood flow in prefrontal brain regions after acute administration to humans. Despite interactions at this level, neuropsychological studies have not found cognitive deficits in abstinent long-term users. Objectives: Here, we wished to investigate the effects of acute ayahuasca intake on neuropsychological performance, specifically on working memory and executive function. Methods: Twenty-four ayahuasca users (11 long-term experienced users and 13 occasional users) were assessed in their habitual setting using the Stroop, Sternberg, and Tower of London tasks prior to and following ayahuasca intake. Results Errors in the Sternberg task increased, whereas reaction times in the Stroop task decreased and accuracy was maintained for the whole sample following ayahuasca intake. Interestingly, results in the Tower of London showed significantly increased execution and resolution times and number of movements for the occasional but not the experienced users. Additionally, a correlation analysis including all subjects showed that impaired performance in the Tower of London was inversely correlated with lifetime ayahuasca use. Conclusions Acute ayahuasca administration impaired working memory but decreased stimulus–response interference. Interestingly, detrimental effects on higher cognition were only observed in the less experienced group. Rather than leading to increased impairment, greater prior exposure to ayahuasca was associated with reduced incapacitation. Compensatory or neuromodulatory effects associated with long-term ayahuasca intake could underlie preserved executive function in experienced users. Keywords: Psychedelics . Ayahuasca . Neuropsychology Executive functions