<p>The current research presents the development of transfersomal hydrogel-based formulation for effective ocular administration of fluconazole, where the hydrogel-base was prepared using hyaluronic acid (HA) and carboxymethyl chitosan (CMCH). Transfersomes loaded with fluconazole were formulated by thin-film hydration and the formulation optimization was performed by a 2³ factorial design-based approach. The optimized formulation (FTD) exhibited 68.55&#xa0;nm vesicle size, 0.250 polydispersity index, – 13.7 mV zeta potential, 71.66 ± 2.15% entrapment efficiency, and 1.009 deformability index. The transmission electron microscopy image exhibited the vesicular morphology of FTD. Afterward, FTD formulation was incorporated within HA/CMCH hydrogel-base to prepare transfersomal hydrogel formulation (FTH), which exhibited 93.18 ± 3.11% drug content, 6.4 ± 0.7 pH, 4.68 × 10³ cPs viscosity (at 10&#xa0;rpm), 36.25 ± 1.25&#xa0;mm spreadability, and 3.85 × 10³ dyne/cm² ex vivo corneal mucoadhesive force. FTH formulation exhibited a spherical to elliptical morphology with smooth surface in Field emission-scanning electron microscopy image. The drug-excipient compatibility within FTH formulation was indicated by Fourier transform-infrared spectroscopy. The results of ex vivo ocular drug permeation exhibited a sustained drug release profile from FTH formulation over 24&#xa0;h. The developed FTH formulation showed significant antifungal activity against <i>Candida albicans</i>. In addition, it was found both stable and non-irritant. Therefore, the findings showed a new approach to improve the ocular fluconazole permeation through HA/CMCH-based transfersomal hydrogel system for effective topical delivery of fluconazole in fungal keratitis management.</p> Graphical Abstract <p></p>