Chagas disease (CD) caused by the protozoanTrypanosoma cruziaffects more than six million people worldwide. Treatment is restricted to benznidazole (Bz) and nifurtimox (Nf) that display low activity in the later chronic stage besides triggering toxic events that result in treatment abandonment. Therefore, new therapeutic options are necessary. In this scenario, natural products emerge as promising alternatives to treat CD. In the family Plumbaginaceae,Plumbagosp. exhibits a broad spectrum of biological and pharmacological activities. Thus, our main objective was to evaluate, in vitro and in silico, the biological effect of crude extracts of root and of aerial parts ofP. auriculata, as well as its naphthoquinone Plumbagin (Pb) againstT. cruzi. The phenotypic assays revealed potent activity of the root extract against different forms (trypomastigote and intracellular forms) and strains (Y and Tulahuen), with a compound concentration that reduced 50% of the number of the parasite (EC50) values ranging from 1.9 to 3.9 µg/mL. In silico analysis showed that Pb is predicted to have good oral absorption and permeability in Caco2 cells, besides excellent probability of absorption by human intestinal cells, without toxic or mutagenic potential effects, not being predicted as a substrate or inhibitor of P-glycoprotein. Pb was as potent as Bz against intracellular forms and displayed a superior trypanosomicidal effect (about 10-fold) in bloodstream forms (EC50= 0.8 µM) as compared to the reference drug (8.5 µM). The cellular targets of Pb onT. cruziwere evaluated using electron microscopy assays and the findings on bloodstream trypomastigotes showed several cellular insults related to the autophagic process. Regarding toxicity in mammalian cells, the root extracts and the naphthoquinone present a moderate toxic profile on fibroblasts and cardiac cell lines. Then, aiming to reduce host toxicity, the root extract and Pb were tested in combination with Bz, and the data showed additive profiles with the sum of the fractional inhibitory concentration indexes (ΣFICIs) being 1.45 and 0.87, respectively. Thus, our work reveals the promising antiparasitic activity ofPlumbago auriculatacrude extracts and its purified naphthoquinone Plumbagin against different forms and strains ofTrypanosoma cruziin vitro.