The development of an effective amphotericin B (AmB) formulation to replace actual treatments available for leishmaniasis, which present serious drawbacks, is a challenge. Here we report the development of hyaluronic acid-amphotericin B self-assembled nanocomplexes (HA-AmB), processed by freeze-drying (FD) or nano spray-drying (SD), using a simple process that favors the non-covalent drug-polysaccharide association in an amorphous state. These water-soluble formulations, which presented a nanometric size (300-600 nm), high colloidal stability (zeta potential around -39 mV) and an AmB loading (15-18%) in aggregated and super aggregated states, demonstrated less in vitro cytotoxic and hemolytic effects compared to the free-drug. A significant decrease in the number of intramacrophagic L. infantum amastigotes upon treatment (IC50 of 0.026 and 0.030 μM for HA-AmB FD and HA-AmB SD, respectively) was also observed, and the best selectivity index (SI) was observed for the HA-AmB SD nanocomplex (SI of 651). Intravenous administration of the HA-AmB SD nanocomplex for 3 alternate days showed an effective parasite reduction in the spleen and liver of C57BL/6 mice without signs of toxicity commonly observed upon free-AmB treatment. Although lower than that achieved with AmBisome® in the liver, the observed parasite reduction for the nanocomplex was of a similar order of magnitude. The efficacy, stability, safety and low cost of the HA-AmB SD nanocomplex highlight its potential as an alternative treatment for leishmaniasis.