The aim of this study was to evaluate the action of homeopathic treatment on mice experimentally infected with Trypanosoma cruzi. Methods: Eighty adult male C57BL/6 inbred mice were randomly allocated to five groups treated with biotherapy (nosode) of T. cruzi 12dH (123) pre- and post-infection; Phosphorus 12dH post-infection; infected control treated with control solution and uninfected control. The biotherapy was prepared by the Costa method from the blood of mice experimentally infected with the Y strain of T. cruzi. Phosphorus was used because of its clinical and reportorial similarity to Chagas disease. T. cruzi (104) sanguineous forms were inoculated intraperitoneally per animal. Parasitaemia was monitored, leukocyte and serological responses were evaluated at 0, 7, 14 and 42 days after infection. The prepatent and patent periods of parasitaemia, maximum of parasitaemia, day of maximum parasitaemia and mortality rates were compared between groups. Results: A significantly shorter period of patent parasitaemia was observed in the group treated with the biotherapy before infection (p < 0.05) than in the other groups. This group also had the lowest parasitaemias values at 9, 13, 15 (p < 0.05), 17 (p < 0.05), 22, 24 and 28 days, a lower rate of mortality and a significant increase of lymphocytes compared to the infected control group. The Phosphorus group had the longest period of patent parasitaemia, higher maximum parasitaemia, and a significant reduction of lymphocyte numbers, but no mortality. The infected control group had the highest mortality rate (not statistically significant), and the highest IgG titres at 42 days post-infection (p < 0.05). Conclusions: The results suggest that pre-treatment with biotherapy modulates host immune response to T. cruzi, mainly during the acute phase of the infection. Phosphorus shows an action on the pathogenicity by T. cruzi infection. Homeopathic treatment of T. cruzi infection should be further investigated.