Ayahuasca has been proposed as a treatment for substance use disorders. However, because of its hallucinogenic properties, studies investigating its abuse potential are needed. The aim of the present study was to investigate voluntary ayahuasca drinking in male mice using a two-bottle choice procedure. Male mice were exposed to two bottles, one of water and one of ayahuasca (0.01, 0.03 or 0.1 mg/ml), for 15 h/day, under 3 protocols (acquisition): (1) every other day access; (2) access every 3 days; (3) access every 5 days. Animals were then submitted to a 14-day drug-free period, followed by 3 re-exposure phases (same conditions as during acquisition), with 7 drug-free days between each. Regardless of the ayahuasca concentration, animals showed a preference for ayahuasca over water when exposed to ayahuasca every other day during the acquisition and re-exposure phases. Extending the period between ayahuasca exposures changed the expression of ayahuasca preference, with the longest break (every 5 days) being associated with preference for water over ayahuasca (i.e., ayahuasca aversion), an effect that was more predominantly observed at higher ayahuasca concentrations. A significant interaction was observed between frequency of exposure to ayahuasca and ayahuasca concentration for total ayahuasca intake during the later re-exposure phase. Our findings show that both the frequency of exposure and the ayahuasca concentration are critical when determining ayahuasca preference in a two-bottle choice model in mice, which can help guide therapeutic/ritualistic ayahuasca use.