The aim of this study was to synthesize polymeric excipients that can form mucoadhesive hydrogels containing amphotericin B (AmB) for the treatment of mucosal leishmaniasis. 2-(2-Acryloylaminoethyldisulfanyl)-nicotinic acid (ACENA) was copolymerized with N-vinyl pyrrolidone to obtain thiolated polyvinylpyrrolidone (PVP) that was then complexed with AmB to improve its solubility. The resulting structure of thiolated PVP was evaluated by H-1 nuclear magnetic resonance to confirm S-protected thiol groups, and the average molecular mass was determined by size exclusion chromatography. Moreover, variants of thiolated PVP-AmB were studied for the thiol content, amount of complexed AmB, cytotoxicity, mucoadhesive properties, and antileishmaniasis activity. The highest achieved degree of thiolation was 772 +/- 24.64 mu mol/g, and the amount of complexed AmB was 27.05 +/- 0.31 mu mol per g of polymer. Thiolated PVP and thiolated PVP-AmB variants (0.5% m/v) showed no cytotoxicity, whereas the equivalent concentration of free AmB reduced Caco-2 cell viability to 70% within 24 h. Thiol-functionalized PVP and PVP-AmB complexes displayed 7.66- and 7.20-fold higher adhesion to the mucosal surface in comparison to unmodified PVP and PVP-AmB, respectively. In addition, variants of thiolated PVP-AmB complexes displayed 100% antileishmaniasis activity in comparison to the 80% killing efficiency of Fungizone, which has been applied in the equivalent AmB concentration of 0.2 mu g/mL. Thiol-functionalized PVP proved to be a promising novel excipient for the delivery of AmB providing enhanced solubility and improved mucoadhesive properties which are beneficial for the treatment of mucosal leishmaniasis.