This article describes the in vivo evaluation of a new topical preparation (Zincostatin) indicated for Candida albicans infected diaper rash. In order to study the influence of the 20% zinc oxide (W/W) (a protectant) on the anti-candidal efficacy of the 100,000 U/g of nystatin found in the ointment, groups of guinea pigs received different treatments with or without local occlusion. Zinc oxide, nystatin, or the combination of both were applied, in the ointment base, on the backs of the animals inoculated with a fresh strain of Candida albicans. Macroscopic and microbiological evaluation of the skin lesions were assessed at regular intervals during 21 days. While the two treatments containing nystatin were more effective than zinc oxide alone, the latter agent did not decrease the efficacy of the antifungal drug in combination therapy. Also, zinc oxide seemed to afford some protection against local maceration induced by occlusion, which increased the severity of the infection.