Ayahuasca, a psychoactive brew originally used ritually and therapeutically by various traditional peoples in the Amazon, is receiving special attention for its potential therapeutic effects on addiction. This study investigates the effects of lyophilized ayahuasca in alcohol-induced animal model, examining behavioral, pharmacological, and neurobiological aspects. Phytochemical analysis confirmed the presence of β-carbolines and dimethyltryptamine, with all compound levels within literature ranges. Pharmacological screening revealed dose-dependent behavioral changes, with the highest dose inducing transient effects. Motor coordination deficits were observed at the highest dose, consistent with pharmacological screening. Pretreatment with ayahuasca prevented ethanol-induced conditioned place preference, suggesting anti-addictive potential. Immunoreactivity analysis revealed no significant changes in DeltaFosB and c-Fos expression in the nucleus accumbens core and shell subsections. This study suggests low acute toxicity of ayahuasca, motor coordination impairment at high doses, and potential anti-addictive effects in preventing ethanol-induced conditioned place preference. These findings contribute to the understanding of the pharmacological and behavioral effects of ayahuasca and highlight the need for further research and clinical studies to elucidate its therapeutic potential in substance use disorders.