Fungal infections pose a significant challenge in numerous developing nations and worldwide, necessitating urgent solutions. Oral administration of antifungal medications often leads to severe adverse reactions. Hence, employing topical delivery systems is preferred to ensure efficient dermal delivery of antifungal agents while minimizing side effects. Furthermore, the incorporation of penetration enhancers into nanocarriers loaded with antifungal agents has demonstrated enhanced efficacy in combating mycotic infections. Consequently, ultra-deformable penetration enhancer-containing vesicles (PEVs) were developed to explore this promising approach. In this study, Labrasol (R) and Transcutol (R) were used as penetration enhancers in formulating ultra-deformable PEVs containing the antifungal agent Fluconazole (FCZ). The PEVs underwent comprehensive characterization, including measurements of particle size (PS), charge, and entrapment efficiency (EE%). The results revealed that the size of tested PEVs ranged from 100 to 762 nm. All particles exhibited a negative charge, with a minimum zeta potential (ZP) of -38.26 mV, and an intermediate entrapment efficiency (EE%) that reached approximately 40%w/w. Ex-vivo studies demonstrated the ability of PEVs to deliver FCZ to the dermis while minimizing transdermal delivery. The selected formula was tested in-vivo using candidiasis-induced rat model and showed a superiority in its antifungal effect against Candida Albicans compared to the drug control. Stability studies were executed for the selected formula, and revealed good stability shown by the insignificant change in the PS, ZP& EE% over a six-month period.