This work aims to evaluate the effect of two natural (whey protein isolate, WPI, and soy lecithin) and a synthetic (Tween 20) emulsifier on physicochemical properties and physical stability of food grade nanoemulsions. Emulsions stabilized by these three surfactants and different sunflower oil contents (30% and 50% w/w), as the dispersed phase, were fabricated at two levels of homogenization pressure (500 and 1000 bar). Nanoemulsions were characterized for droplet size distribution, Zeta-potential, rheological properties, and physical stability. Dynamic light scattering showed that droplet size distributions and D50 values were strongly affected by the surfactant used and the oil content. WPI gave similar droplet diameters to Tween 20 and soy lecithin gave the larger diameters. The rheology of emulsions presented a Newtonian behavior, except for WPI-stabilized emulsions at 50% of oil, presenting a shear-thinning behavior. The physical stability of the emulsions depended on the surfactant used, with increasing order of stability as follows: soy lecithin < Tween 20 < WPI. From our results, we conclude that WPI is an effective natural replacement of synthetic surfactant (Tween 20) for the fabrication of food-grade nanoemulsions.