Carvacrol is present abundantly in the essential oils of many medicinal plants and well known for its numerous biological activities. Since partial solubility in water and physicochemical instability limits its industrial uses, the present study was performed to prepare a carvacrol nanoemulsion (CANE) using an ultrasonication technique and further evaluation of its anticancer potential against human lung adenocarcinoma A549 cells. The nanoemulsion formulation was optimized by varying carvacrol and polysorbate 80 ratios and characterized by dynamic light scattering (DLS), which revealed a negative surface charge with a mean droplet size between 105.5 ± 3.4 to 169.8 ± 4.9 nm. The CANE induced reactive oxygen species (ROS) production in A549 cells, leading to activation of key regulators of apoptosis such as p-JNK, Bax and Bcl2 as well as release of cytochrome C, and activation of the caspase cascade. Suppression of mitochondrial ROS using Mito-TEMPO reversed the apoptotic potential of CANE signifying involvement of mitochondrial ROS in cell death. Beside, CANE displayed a strong antitumor potential in vivo using an athymic nude mice model. The results strongly support that CANE induced apoptosis in A549 cells by induction of ROS and could be a promising candidate for lung cancer therapy.