Amphotericin B (AmB) is a highly hydrophobic drug with significant leishmanicidal activity whose use is limited by its poor water solubility and adverse effects. Polymer-drug conjugates are proposed as a delivery system designed to overcome those limitations while improving drug bioavailability, safety, and activity. Here, AmB was covalently linked to periodate-oxidized hyaluronic acid (HA) (oxidation degree of 30.1 ± 5.6%) via a Schiff base (HA-AmB imine). The conjugate presents high water solubility and self-assembles into particles with a mean size of 88.2 ± 17.6 nm, a negative charge (-28.3 ± 0.9 mV), and a drug content of 17.8 ± 1.4%. Spectroscopic studies revealed the presence of AmB in aggregate and super-aggregated forms in the conjugate, which could explain the significant reduction of the in vitro cytotoxicity and hemolytic activity. The formulation showed not only in vitro anti-leishmanial activity against L. infantum-infected macrophages (IC50 = 0.023 μM) but also against an in vivo infected mouse model, promoting a 1.32- and a 4.98-log10 suppression of the L. infantum burden in the spleens and liver, respectively, without toxic effects. In summary, this study describes the safe and effective use of water-soluble HA-AmB imine conjugates for leishmaniasis treatment.