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Although Nystatin has been used since 1950s as a non-absorbable antifungal agent, there is still no reliable in-vivo data available stating a dose–effect relationship of Nystatin-suspension in the treatment of oropharyngeal infection with Candida albicans. Here, we studied the efficacy of a commercially available topical Nystatin suspension in a new ex-vivo model of candidiasis using porcine oral mucosa. After 48 and 96 h of C. albicans infection, 230 IU Nystatin (standard dosage), 100 IU and 20 IU proved to be equally efficacious. Multiple applications of Nystatin were not superior compared with single application. In dosages of 10 and 0.1 IU the activity of Nystatin suspension against C. albicans was no longer confirmed. In an agar diffusion model, the minimal biocidal concentration of Nystatin proved to be 0.25 IU. Our results suggest that the proposed porcine ex-vivo model is much closer to the in-vivo situation compared with other established in-vitro models of the treatment of muco-cutaneous candidiasis and may provide a substitute for animal models in the investigation of antifungal agents. Additionally, it seems to be a valuable tool for further investigations of the pathogenesis of C. albicans infections.