Fungal eye infections are largely disseminated, especially in developing countries where they may leave over half a million people blind per year. The current study aims to boost the voriconazole antifungal efficiency via loading it as cubosomes (VZ-Cub) into hyaluronic acid and poloxamer-based ocular in situ gel. VZ-Cub were fabricated applying Box-Behnken design and employing phytantriol, poloxamer F127, and VZ amounts as independent variables. The produced nano vesicles were evaluated for the dependent variables of particle size (PS), entrapment efficiency (EE%), and transcorneal steady-state flux (Jss) of the VZ, and, the obtained optimal VZ-Cub was loaded into an in situ gel base to enhance its ocular residence time. The in situ gel formulation was tested for its gelation temperature, drug release behavior, transcorneal permeation effects, and antifungal activity. The optimized VZ-Cub consisted of 100 mg of phytantriol, 60 mg of poloxamer F127, and 21 mg of VZ. This formulation led to a minimum PS of 71 nm, an EE% of 66%, Jss value of 6.5 µg/(cm2·min), and stability index of 94 ± 2%. The optimized VZ-Cub-loaded in situ gel released 84% VZ after 12 h and yielded a 4.5-fold increase in drug permeation compared with the VZ aqueous dispersion. The antifungal activity, which was obtained by measuring the fungal growth inhibition zones, revealed that the VZ-Cub-loaded in situ gel formulation had a 3.89-fold increase in antifungal activity compared with the VZ dispersion. In summary, an ocular in situ gel loaded with VZ-Cub could be an effective novel nano-paradigm with enhanced transcorneal permeation and antifungal properties.