Vaginal candidiasis is a medical condition that affects large majority of the women at least once in their lifetime. The condition manifests with itching, irritation, and discharges which is troublesome for women during mundane activities. The purpose of this research was to formulate and evaluate the physicochemical properties and drug permeation of Fenticonazole-loaded cubogel through vaginal mucosa. Concisely, the drug-loaded cubosomes were prepared via hot dispersion emulsification technique. Following, the percent drug entrapment efficiency, particle size, polydispersity index, and zeta potential of the cubosomes were determined. Optimization criteria involved maximizing entrapment efficiency (EE %) and zeta potential (ZP), while maintaining a nanoscale particle size to ensure colloidal stability. The optimized formulation exhibited a high desirability score of 0.933 with EE % of 85.32 __-mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"-__± 2.34 %, PS of 169 __-mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"-__± 0.85 nm, PDI of 0.29 __-mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"-__± 0.02, and ZP of -24.40 __-mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"-__± 1.27 mV. In addition, 86.77 __-mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"-__± 3.79 % of Fenticonazole nitrate was released from the optimum cubosomal formulation after 8 h. Cubical nanovesicles were revealed via transmission electron microscope while infrared spectroscopy revealed the lack of interaction between the used components. Stability was unchanged upon storage for three months. The rheogram of the optimum formulation-loaded cubogel suggested a shear-thinning behavior. Additionally, the optimum cubogel demonstrated higher biofilm inhibitory effect compared to the drug suspension. Similarly, both, ex vivo permeation and confocal laser scanning, suggested the enhanced vaginal epithelium permeability and the deeper vaginal mucosa penetration of the optimum cubogel, compared to the drug suspension and aqueous Rhodamine B carbopol gel, respectively. Histopathological assessment concluded with the safety of the cubogel on the vaginal mucosal epithelium and underlying tissue.