Rosemary essential oil (REO) is a natural phenolic compound with high antimicrobial activity. However, its application is limited due to the poor solubility in water, instability, and high volatility. In this study, REO is incorporated into the high amylose corn starch (HACS) nanostructures by inclusion complexation. The characterization of the obtained nanostructures is done by SEM, TGA, FTIR, and XRD. Encapsulation efficiency, loading capacity, stability, release, and antibacterial activity of encapsulated REO are analyzed. The nanostructures show spherical nanoparticles with an average size lower than 200 nm. The results of XRD and FTIR confirm the incorporation of REO into the amylose nanostructures. Thermogravimetric analysis indicates that the inclusion complex of REO and HACS increases the thermal stability of REO significantly. The solubility and antibacterial activity of REO are enhanced after complexation with amylose nanostructures. Minimum inhibitory concentration (MIC) of free REO is 3.52 mg mL−1 or 0.39% v/v and for encapsulated REO is decreased to 2.5 mg mL−1. The results suggest that HACS can be used as a potential carrier for sensitive and hydrophobic compounds to enhance their application in the pharmaceutical and food industries.