Purpose The aim of this study was to elaborate new forms of biofungicide formulations which could increase biological activity of essential oil against various strains of pathogenic fungi of plants, dermatophytes, and molds. Methods The nanoemulsions containing four various essential oils (cinnamon, thyme, manuka, and tea tree oil) were obtained by using the low-energy (PIC) and the high-energy emulsification methods (ultrasonification). The physicochemical properties and activity of prepared systems against strains of pathogenic fungi of plants (F. culmorum, Ph. cactorum), dermatophytes (T. mentagrophytes M. gypseum) and molds (S. brevicaulis, A. niger) were examined. Fungicidal activity was tested by the method of linear growth of mycelium on an agar medium. Macroemulsions containing the oils and the pure essential oils were used as comparative samples.Results It was found that nanoemulsions prepared by ultrasonification showed excellent fungicidal activity compared to pure oils and macroemulsions. Among others, the manuka oil nanoformulations showed the highest activity against the tested fungi. Conclusions Nanoemulsions can be applied as the effective carriers of essential oils. They allow the reduction of the concentration of the bioactive oils while maintaining biological activity. The obtained nanosystems can be applied as safe, biodegradable, eco-friendly antifungal products in pharmaceutical, cosmetic, and agrochemical industries as they increase the biological activity of the tested oils against various type of fungi.