Although the tobacco plant has been employed as a medicinal and sacred herb by Indigenous cultures across the Americas, its usage drastically changed after the 15th-century colonial arrival; its large-scale commodification and global marketing once brought to Europe lead to hedonic and addictive uses harmful to health. As a consequence, tobacco smoking is now one of the largest public health problems worldwide. However, in the Peruvian Amazon, a region of origin of tobacco species, Indigenous healers still know how to use the plant for therapeutic purposes. Due to a general disregard of Indigenous knowledge and stigma, these uses have however not so far been clinically investigated. We hence conducted for the first time a clinical field study assessing a sample of patients treated by a traditional healer specialized in tobacco in the Peruvian Amazon (observational design, pilot study, N = 27). The study was conducted within a transdisciplinary and multi-epistemic medical frame, in close partnership with an Amazonian healer. We used validated self-report scales to quantitatively assess mental health variables before and after the weeklong treatment, and mixed-methods to report experienced effects. Paired-samples t-tests comparing pre- and post-treatment scores revealed significant reductions in anxiety, depression, perceived stress, and general symptom indicators. Experienced effects included initial physical discomfort, followed by psychologically or existentially/spiritually significant insights. Our findings point to a sophisticated therapeutic approach based on Indigenous knowledge of tobacco applications, which should be further investigated. The study also contributes to the burgeoning scientific field on therapeutic uses of contentious psychoactive plants.