Ayahuasca is a beverage used in religious rituals of indigenous and nonindigenous groups, and its therapeutic potential has been investigated. Ayahuasca is obtained by decoction of the Banisteriopsis caapi that contains β-carbolines (harmine, harmaline, and tetrahydroharmine) plus Psychotria viridis that contains N,N-dimethyltryptamine. Although plants used in folk medicine are recognized as safe, many of them have genotoxic potential. The Salmonella/microsome assay is usually the first line of the mutagenicity evaluation of products intended for therapeutic use. Our objective was to evaluate the mutagenicity of ayahuasca beverage and their constituents using the Salmonella/microsome assay with TA98 and TA100. We analyzed two ayahuasca samples, and also beverage samples prepared each individual plant P. viridis and B. caapi. Harmine and harmaline were also tested. All beverage samples were chemically characterized and both ayahuasca samples could be considered representative of the beverages consumed in religious rituals. Both ayahuasca samples were mutagenic for TA98 and TA100 with and without S9, with similar potencies. The beverage obtained from P. viridis was not mutagenic, and beverage obtained from B. caapi was mutagenic for TA98 with and without S9. Harmine was nonmutagenic and harmaline was mutagenic only for TA98 without S9. Harmaline fully explain the mutagenicity observed with TA98 without S9 of both ayahuasca samples and the B. caapi beverage samples. We conclude that the ayahuasca samples are mutagenic and this effect is partially explained by harmaline, one of the β-carbolines present in the beverage. Other mutagenic compounds seem to be present and need to be further investigated.