Abstract Benznidazole (Bz) is the recommended drug for the treatment of Chagas disease; however, its efficacy may vary according to the sensitivity of Trypanosoma cruzi strains to the drug and host immune background. The study evaluated the immune response of peripheral blood mononuclear cells (PBMC) that were infected in vitro with the Colombian strain (Col) and treated with Bz. The co‐cultures were incubated for 24 h, 5 and 10 days, where cytokine dosage was performed in the supernatant and evaluation of the cells for CD28 + and CTLA‐4 + molecules in CD4 + and CD8 + lymphocytes, and CD80 + , CD86 + and HLA‐DR + in CD14 + cells. The results showed that Col induced a strong inflammatory response, with an increase in IFN‐γ and TNF early in the infection (24 h), however, from 5 days of infection on, TNF production declined, and IL‐10 production increased, which may be associated with a control mechanism of the exacerbated inflammatory response. The Bz treatment did not significantly alter the frequencies of the phenotypes evaluated both T cell subsets and CD14 + cells. Therefore, this study reinforces the need for typing the patient's strain to guide therapy and promote individualized treatment protocols due to the heterogeneous genetic background among T. cruzi strains.