Background: The evaluation of antifungal agents by in vitro and animal experiments cannot predict clinical efficacy with certainty. New models are needed to assess and compare antifungal activity. Objective: We compared on human stratum corneum ex vivo the antifungal activity and lingering effect of 200 mg itraconazole daily and twice daily, and 250 mg/day terbinafine. Methods: Three groups of 10 healthy volunteers entered the open comparative trial. Results were evaluated in a blinded manner. Cyanoacrylate skin surface strippings (CSSS) were taken from the back and superficial dermatome skin samples (SDSS) were taken from plantar skin at days 0, 1, 3, 7, 8, 10, 14, 21, 28, and 35. Spores or yeasts of selected fungi ( Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis, and Candida albicans) were deposited and cultured on the CSSS and SDSS. The 1-week fungal growth on CSSS and SDSS was assessed over time by computerized image analysis to derive the inhibitory effect of the oral antifungal agents administered. Fungitoxic activity was also assessed by the use of 2-day cultures on CSSS followed by a transfer to Sabouraud medium. Results: Comparable antifungal activity against dermatophytes was found for all three regimens. Itraconazole at both dosages was always significantly more active than terbinafine against C. albicans on CSSS and SDSS. Overall, 200 mg itraconazole twice daily appeared to be more fungitoxic than 250 mg/day terbinafine and 200 mg/day itraconazole. Conclusion: The ex vivo culture of fungi on human stratum corneum is very similar to the in vivo situation. Both itraconazole and terbinafine display high antidermatophyte activity. Faster onset and longer posttherapy activity were demonstrated in the itraconazole treatment groups. Terbinafine had marginal activity against C. albicans in this model.