Chagas disease is a chronic infection caused by Trypanosoma cruzi. The first-line drugs approved for treatment have several limitations and are associated with toxicity. The recombinant TSA-1-C4 and Tc24-C4 proteins have been profiled as promising candidates for the formulation of therapeutic vaccines, leading us to propose them in combination as a bivalent recombinant protein strategy. In this study, we evaluated the immunomodulatory effect of TSA-1-C4 plus Tc24-C4 recombinant protein combination by in-vitro assays. Macrophages from naïve BALB/c mice were isolated and stimulated with TSA-1-C4+Tc24-C4 proteins, supernatants were recovered to measure NO, H O , and, TNF-α, IL-1β, IL-6 and IL-10 cytokines. Stimulated macrophages were co-cultured with CD8 T cells from naïve mice, and inflammatory cytokine-profile was measured from supernatants. Additionally, by experimental T. cruzi acute infection model, cytotoxicity was evaluated in mice after the treatment with TSA-1-C4+Tc24-C4 proteins in presence of a TLR-4 agonist adjuvant, E6020-SE. We observed that the bivalent recombinant protein strategy activates macrophages by NO and H O production, also induced a significant Th1 immune-response compared to either TSA-1-C4 or Tc24-C4 stimulated macrophages. Moreover, naïve CD8 T cells in presence of TSA-1-C4+Tc24-C4 stimulated-macrophages similarly boosted Th1 profile by significant production of IFN-γ and TNF-α cytokines. These results support the synergistic effect of the bivalent recombinant protein strategy, which leads activation of peritoneal macrophages and CD8 T cells eliciting the Th1 immune response. Although the administration of TSA-1-C4+Tc24-C4+E6020-SE showed cytotoxic activity in T. cruzi-infected mice, there was not a benefit compared to stimulate with TSA-1-C4 or Tc24-C4 antigens formulated with E6020-SE adjuvant.