Ethnopharmacological relevance: Ayahuasca (AYA) is a psychedelic brew used in religious ceremonies. It is broadly used as a sacred medicine for treating several ailments, including pain of various origins. Aim of the study: To investigate the antinociceptive effects of AYA and its mechanisms in preclinical models of acute and chronic pain in mice, in particular during experimental neuropathy. Materials and methods: The antinociceptive effects of AYA administered orally were assessed in the following models of pain: formalin test, Complete Freund's Adjuvant (CFA)-induced inflammation, tail flick test, and partial sciatic nerve ligation model of neuropathic pain. Antagonism assays and Fos immunohistochemistry in the brain were performed. AYA-induced toxicity was investigated. AYA was chemically characterized. The antinociceptive effect of harmine, the major component present in AYA, was investigated. Results: AYA (24–3000 μL/kg) dose-dependently reduced formalin-induced pain-like behaviors and CFA-induced mechanical allodynia but did not affect CFA-induced paw edema or tail flick latency. During experimental neuropathy, single treatments with AYA (24–3000 μL/kg) reduced mechanical allodynia; daily treatments once or twice a day for 14 days promoted consistent and sustained antinociception. The antinociceptive effect of AYA (600 μL/kg) was reverted by bicuculline (1 mg/kg) and methysergide (5 mg/kg), but not by naloxone (5 mg/kg), phaclofen (2 mg/kg), and rimonabant (10 mg/kg), suggesting the roles of GABAA and serotonergic receptors. AYA increased Fos expression in the ventrolateral periaqueductal gray and nucleus raphe magnus after 1 h, but not after 6 h or 14 days of daily treatments. AYA (600 μL/kg) twice a day for 14 days did not alter mice's motor function, spontaneous locomotion, body weight, food and water intake, hematological, biochemical, and histopathological parameters. Harmine (3.5 mg/kg) promoted consistent antinociception during experimental neuropathy. Conclusions: AYA promotes consistent antinociceptive effects in different mouse models of pain without inducing detectable toxic effects. Harmine is at least partially accountable for the antinociceptive properties of AYA.