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This study aimed to prepare anti-<i>Staphylococcus aureus</i> inclusion complexes (ICs) of Hinoki essential oil (HEO) with β-cyclodextrin (β-CD) and 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD). An ultrasound-assisted kneading method was applied for the complexation for the first time. The recovery yield, embedding fraction and loading capacity of the HEO/β-CD ICs were 92.5%, 78.0% and 11.9%, respectively, while the corresponding values were 80.8%, 73.7% and 12.9% for the HEO/2-HP-β-CD ICs. As well, a comparative study confirmed the efficiency of the ultrasound-assisted kneading method was higher than the traditional kneading method. The results of SEM, XRD, GC-MS and FT-IR suggested the successful formation of ICs. A significant anti-<i>Staphylococcus aureus</i> activity of the fabricated ICs was demonstrated using a colony counting method. Notably, when the dose in liquid culture medium was 20 g L⁻¹, inhibitory rates of 99.8% for HEO/β-CD ICs and 100% for HEO/2-HP-β-CD ICs were achieved. Furthermore, the hydrophilic property of the ICs was proved by water contact angle measurements, implying they have the potential to act as anti-<i>Staphylococcus aureus</i> agents for blending with hydrophilic biodegradable materials for diverse food packaging utilizations.