ABSTRACT The objective of this study was to evaluate the antifungal activity of nanoemulsions encapsulating essential oil of oregano (Origanum vulgare), both in vitro and after application on Minas Padrão cheese. Nanodispersions were obtained by the phase inversion temperature method. Cladosporium sp., Fusarium sp., and Penicillium sp. genera were isolated from cheese samples and used to evaluate antifungal activity. Minimal inhibitory concentrations of non-encapsulated and encapsulated oregano essential oil were determined, and they were influenced by the encapsulation of the essential oil depending on the type of fungus. The antifungal activity of the nanoencapsulated oregano essential oil in cheese slices showed no evidence of an effect of the MICs, when applied in the matrix. On the other hand, an influence of contact time of the nanoemulsion with the cheese was observed, due to the increase in water activity. It was concluded that nanoencapsulated oregano essential oil presented an inhibitory effect against the three genera of fungi evaluated. If environmental parameters, such as storage temperature and water activity, were controlled, the inhibitory effect of nanoemulsions of oregano oil could possibly be greatly improved, and they could be presented as a potential alternative for the preservation of Minas Padrão cheese against fungal contamination.