Ayahuasca is a hallucinogenic infusion used in religious rituals that has serotoninergic properties and may be a potential therapeutic option for drug addiction. In this study, Wistar rats had intermittent access to ethanol for 8 weeks, receiving water (control), naltrexone (NTX, 2 mg/kg body weight [bw] intraperitoneally [i.p.]) or ayahuasca (Aya) at 0.5x, 1x, or 2x the ritual dose in the final 5 days. A naïve group had access only to water. Ethanol intake was estimated throughout the experiment, and cFos expression was evaluated in medial orbital cortex (MO), ventral orbital cortex (VO), lateral orbital cortex (LO), nucleus accumbens (NAc), and striatum. Treatment with either NTX or Aya (oral) did not decrease ethanol intake compared to the baseline level (5th to 7th week), but the NTX group intake was significantly lower than controls (p < 0.05). Ethanol significantly increased cFos expression in the MO region for control (p < 0.0001), NTX (p < 0.05), Aya1 (p < 0.001), and Aya2 (p < 0.0001) groups. This increase was also observed in the VO for the Aya1 group (p ¼ 0.035), in the LO for the Aya2 group (p < 0.01), and in NAc for NTX and ayahuasca groups (p < 0.005). Furthermore, NTX and Aya0.5 treatment decreased cFos expression compared to controls in the MO region (p < 0.05 and p < 0.01, respectively), but only the ayahuasca group reached levels not significantly different from the naïve group. Studies using other protocols and dose regime are necessary to better investigate the impact of ayahuasca on alcohol intake by rats to support the observations in humans. Additionally, the role of ayahuasca in mediating cFos expression in other selected brain regions and its relationship with the serotoninergic/dopaminergic systems and drug addiction need further investigation.