AbstractNanoencapsulation is an attractive novel technique used for incorporating essential oils in food preparations and pharmaceutical formulae. This study investigated the effect of nanoencapsulation on the composition of volatile compounds, as well as the antioxidant and anticancer activities of hydrodistilled (HD) Origanum glandulosum Desf. Oil, which was encapsulated into nanocapsules via High Speed Homogenization (HSH) and into nanoemulsions through High Pressure Homogenization (HPH). Thirty-two volatile components were identified using Gas Chromatography-Mass Spectrometry analysis (GC-MS) in HD essential oil representing 99.04% of the total oil content. GC-MS analysis showed that the use of HPH to prepare nanoemulsions negatively affected the active compounds present in HD oil, particularly carvacrol and thymol, whereas the use of HSH led to significant quantitative differences in the composition of volatiles between HD oil and nanocapsules but generated the same profile. Consistent with the differences in total phenolics, total flavonoids, and volatiles identified in HD and nanoparticles, HD essential oil exhibited a higher antioxidant activity (IC₅₀ 4.22 mg/mL) than nanocapsules (IC₅₀ 57.51 mg/mL) and nanoemulsion (IC₅₀ 78.50 mg/mL), while nanocapsules showed the strongest cytotoxic effect on liver cancer cell line Hep-G2 (54.93 μg/mL) in comparison to HD oil (73.13 μg/mL) and nanoemulsions (131.6 μg/mL).