This study used a murine model of Chagas disease to investigate the isolated and combined impact of Trypanosoma cruzi infection and benznidazole (BZ) therapy on liver structure and function. Male C57BL/6 mice were challenged with T. cruzi and BZ for 15 days. Serum levels of cytokines and hepatic enzymes, liver oxidative stress, morphology, collagen, and glycogen content were monitored. Separately, T. cruzi infection and BZ treatment resulted in a pro-oxidant status and hepatic reactive damage. Concurrently, both T. cruzi infection and BZ treatment induced upregulation of antioxidant enzymes and pathological reorganization of the liver parenchyma and stroma. T. cruzi infection increased serum levels of Th1 cytokines, which were reduced by BZ in both infected and non-infected animals. BZ also induced functional organ damage, increasing serum levels of liver enzymes. When combined, T. cruzi infection and BZ therapy elicited intense hepatic reactive damage that was not compensated by antioxidant enzymatic reaction, subsequently culminating in more severe morphofunctional hepatic injury. Taken together, these findings indicate that during specific treatment of Chagas disease, hepatic pathology may be a result of an interaction between BZ metabolism and specific mechanisms activated during the natural course of T. cruzi infection, rather than an isolated toxic effect of BZ on liver structure and function.