Objectives: Clotrimazole is a broad-spectrum antimycotic that widely used to treat fungal infections topically. However, poor water solubility of clotrimazole and low skin retention of its topical products present a hindrance for local availability and effectiveness of this drug. This study aimed to formulate and evaluate cubosomes as skin retentive system for topical delivery of clotrimazole. Methods: Six clotrimazole cubosomal dispersions (F1-F6) were prepared by emulsification of glyceryl monoloeate (GMO) and poloxamer 407 at different compositions in water with and without polyvinyl alcohol (PVA).The dispersions were examined under polarizing microscope and transmission electron microscope (TEM) for confirming cubosomes formation. The four cubosomal dispersions (F3- F6) that found to be successfully developed were evaluated in terms of pH, particle size, zeta potential, rheological behavior, entrapment efficiency and in vitro drug release. The formulae F3 (without PVA) and F6 (with PVA) comprising  the highest concentration of poloxamer in the disperse phase (15%w/w) provided the highest clotrimazole cumulative percentage release and subjected to comparative ex vivo skin retention and permeation studies with marketed clotrimazole cream. Results: Both F3and F6 showed significantly lower clotrimazole permeation compared to the cream. Moreover, F6achieved the highest skin retention for clotrimazole. The F6 was compared with the cream for its irritation potential in rats and also for antifungal activity in rats inducted with candida albicans. The investigations showed that F6 was well tolerated as the cream. Also,F6 showed superior antifungal activity compared to the available marketed cream. Conclusions: The developed F6 cubosomes is a promising dosage form for safe and effective topical delivery of clotrimazole.