Chagas disease is a neglected endemic disease prevalent in Latin American countries, affecting around 8 million people. The first-line treatment, benznidazole (BNZ), is effective in the acute stage of the disease but has limited efficacy in the chronic stage, possibly because current treatment regimens do not eradicate transiently dormant Trypanosoma cruzi amastigotes. Nanostructured lipid carriers (NLC) appear to be a promising approach for delivering pharmaceutical active ingredients as they can have a positive impact on bioavailability by modifying the absorption, distribution, and elimination of the drug. In this study, BNZ was successfully loaded into nanocarriers composed of myristyl myristate/Crodamol oil/poloxamer 188 prepared by ultrasonication. A stable NLC formulation was obtained, with ≈80% encapsulation efficiency (%EE) and a biphasic drug release profile with an initial burst release followed by a prolonged phase. The hydrodynamic average diameter and zeta potential of NLC obtained by dynamic light scattering were approximately 150 nm and -13 mV, respectively, while spherical and well-distributed nanoparticles were observed by transmission electron microscopy. Fourier-transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis, and small-angle X-ray scattering analyses of the nanoparticles indicated that BNZ might be dispersed in the nanoparticle matrix in an amorphous state. The mean size, zeta potential, polydispersity index, and %EE of the formulation remained stable for at least six months. The hemolytic effect of the nanoparticles was insignificant compared to that of the positive lysis control. The nanoparticle formulation exhibited similar performance in vitro against T. cruzi compared to free BNZ. No formulation-related cytotoxic effects were observed on either Vero or CHO cells. Moreover, BNZ showed a 50% reduction in CHO cell viability at 125 µg/mL, whereas NLC-BNZ and non-loaded NLC did not exert a significant effect on cell viability at the same concentration. These results show potential for the development of new nanomedicines against T. cruzi.