Disability and mortality as consequence of Chagas disease is enormous in South America. Recently, the success of the trypanocidal treatment with benznidazole, the only available drug, has been associated with the host immune response. In the current study, the impact of benznidazole administration immediately after the experimental infection with Trypanosoma cruzi was evaluated in the main lymphocyte populations in lymphoid organs. Untreated mice displayed enlargement of spleen and lymph node related to the increased frequency of T and B lymphocytes, respectively. An intense thymus involution with the depletion of CD4(+)CD8(+) double-positive thymocytes also occurred. Benznidazole treatment led to a partial reversion of the spleen and lymph node enlargement related to changes in the frequency of lymphocyte subsets due to infection. Prevention of thymus involution was achieved, with the profile of thymocyte subsets similar to that of non-infected mice. The parasitic load at the onset of T. cruzi infection seems critical to trigger immune system activation.