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Plasticizers are employed to stabilize films by safeguarding their physical stability and avoiding the degradation of the loaded therapeutic drug during processing and storage. In the present study, the plasticizer effect (glycerol) was studied on bioadhesive films based on sodium alginate (SA), carboxymethyl cellulose (CMC) and gelatin (GE) polymers loaded with amphotericin B (AmB). The main objective of the current study was to assess the morphological, mechanical, thermal, optical, and barrier properties of the films as a function of glycerol (Gly) concentration (0.5–1.5 %) using different techniques such as Scanning Electron Microscope (SEM), Texture analyzer (TA), Differential Scanning Calorimeter (DSC), X-Ray Diffraction (XRD), and Fourier Transforms Infrared Spectroscopy (FTIR). The concentration increase of glycerol resulted in an increase in Water Vapor Permeability (WVP) (0.187–0.334), elongation at break (EAB) (0.88–35.48 %), thickness (0.032–0.065 mm) and moisture level (17.5–41.76 %) whereas opacity, tensile strength (TS) (16.81–0.86 MPa), and young's modulus (YM) (0.194–0.002 MPa) values decreased. Glycerol incorporation in the film-Forming solution decreased the brittleness and fragility of the films. Fourier Transform Infrared (FTIR) spectra showed that intermolecular hydrogen bonding occurred between glycerol and polymers in plasticized films compared to control films. Furthermore, molecular docking was applied to predict the binding interactions betweem AmB, CMC, gelatin, SA and glycerol, which further endorsed the stabilizing effects of glycerol in the complex formation between AmB, CMC, SA, and gelatin. The Findings of the current study demonstrated that this polymeric blend could be used to successfully prepare bioadhesive films with glycerol as a plasticizer.