We have investigated the influences of gamma interferon (IFN-γ) and interleukin-12 (IL-12) on the efficacy of posaconazole (POS) treatment of acute experimental infections with Trypanosoma cruzi; the standard drug, benznidazole (BZ), was used as a positive control. Wild-type (WT) mice infected with T. cruzi and treated with POS or BZ had no parasitemia, 100% survival, and cure rates of 86 to 89%. IFN-γ-knockout (KO) mice infected with T. cruzi and treated with BZ controlled the infection during treatment but relapsed after the drug pressure ceased and had 0% survival, while those receiving POS better controlled the infection after the end of treatment and had 70% survival (P < 0.0001 compared to the results for both untreated and BZ-treated animals). IL-12-KO mice infected and treated with POS or BZ had intermediate results, displaying enhanced parasitemia, decreased survival (77 to 83%), and reduced cure rates (35 to 39%) compared with those of the WT animals. Our results demonstrate that either IFN-γ or IL-12 deficiency reduces the efficacy of POS or BZ in this experimental model but also indicate that the anti-T. cruzi activity of POS is much less dependent on the activity of IFN-γ than that of BZ is.