The food industry has increased its interest in using "consumer-friendly" and natural ingredients to produce food products. In the case of emulsifiers, one of the possibilities is to use biopolymers with emulsification capacity, such as octenyl succinic anhydride modified starch, which can be used in combination with other polysaccharides, such as chitosan and carboxymethylcellulose, in order to improve the capacity to protect bioactive compounds. In this work, multilayer nano-emulsion systems loaded with oregano essential oil were produced by high energy methods and characterized. The process optimization was carried out based on the evaluation of particle size, polydispersity index, and zeta potential. Optimal conditions were achieved for one-layer nano-emulsions resulting in particle size and zeta potential of 180 nm and -42 mV, two layers (after chitosan addition) at 226 nm and 35 mV, and three layers (after carboxymethylcellulose addition) of 265 nm and -1 mV, respectively. The encapsulation efficiency of oregano essential oil within nano-emulsions was 97.1%. Stability was evaluated up to 21 days at 4 and 20 °C. The three layers nano-emulsion demonstrated to be an efficient delivery system of oregano essential oil, making 40% of the initial oregano essential oil available versus 13% obtained for oregano essential oil in oil, after exposure to simulated digestive conditions.