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Cardamom essential oil (CEO) loaded food grade nanostructured lipid carriers (NLCs) were produced using cocoa butter (as solid lipid) and Tween 80 (as surfactant) for application in aqueous-based foods. The results demonstrated that the developed NLCs had fine size (<150 nm) and high entrapment efficiency (>90%). Differential scanning calorimetry (DSC) results showed a less-ordered crystalline structure leading to high loading capacity. All samples exhibited perfect type crystal (or β-modification crystal) and a less crystalline state in X-ray diffractograms. Few changes were detected in the turbidity of systems after storage time, demonstrating that the NLCs did not show significant aggregation. Antimicrobial activity of CEO emulsion and CEO loaded NLC formulations were investigated by Broth Macrodilution method and confirmed that, encapsulation was able to protect the antimicrobial activity of CEO. The results showed that CEO-loaded NLC could be used as food supplements.