The influence of ionic surfactants (cationic surfactant lauric arginate and anionic surfactant lysolecithin) on the physical properties, antifungal and mycotoxin inhibitory efficacy of Tween 80 stabilized cinnamon oil-in-water nanoemulsions was investigated. Nanoemulsion droplets of similar particle diameter (∼100 nm), but variable electrical characteristics, were formed by mixing 0.1 wt% ionic surfactant with 0.9 wt% Tween 80 before homogenization. The nanoemulsions were physically stable over 28 days at 23 °C. The antifungal activity (against mycelial growth and spore germination) and mycotoxin inhibitory activity of cinnamon oil nanoemulsions bearing positive, neutral, and negative charge surface was then evaluated against two chemotypes of Fusarium graminearum. In general, the cinnamon oil played a decisive role in the resulting antifungal and mycotoxin inhibitory activities. The surfactant charge had a limited impact on the antifungal mycotoxin inhibitory activities of cinnamon oil in the nanoemulsions. Both ionic surfactant-based cinnamon oil nanoemulsions showed greater activity in inhibiting mycelial growth and mycotoxin production of F. graminearum than those based on Tween 80. Treatment of mycelium with cinnamon oil nanoemulsions resulted in the loss of cytoplasm from fungal hyphae, and accounted for the antifungal action. These results have important implications for the design of essential oil based nanoemulsions as effective antifungal delivery systems in foods.