In this study, we report that the essential oil obtained from Curcuma zedoaria Roscoe, known as zedoary, possesses efficient cytotoxic effects on non-small cell lung carcinoma (NSCLC) cells and causes cell apoptosis. Zedoary essential oil increased the sub-G1 population and the level of annexin-V binding and induced cleavage and activation of caspase-3, -8, and -9 and poly(ADP ribose) polymerase. Decreases in the levels of Bcl-2 and Bcl-xL and an increase in the Bax/Bcl-2 ratio were also observed following zedoary essential oil treatment. Notably, zedoary essential oil led to the release of AIF, endonuclease G, and cytochrome c into the cytosol and increased levels of p53 in H1299 cells. Our results indicate that zedoary essential oil slightly inhibited the phosphorylation of ERK1/2 and enhanced the phosphorylation of JNK1/2 and p38. Zedoary essential oil also inhibited AKT/NF-κB signaling pathways in H1299 cells. Moreover, intraperitoneal administration of zedoary essential oil significantly suppressed the growth of H1299 cells in vivo. In addition, potential active compounds were detected using gas chromatography and mass spectrometry. 8,9-Dehydro-9-formyl-cycloisolongifolene, 6-ethenyl-4,5,6,7-tetrahydro-3,6-dimethyl-5-isopropenyl-trans-benzofuran, eucalyptol, and γ-elemene were found in zedoary essential oil. In summary, our findings provide insight into the molecular mechanisms underlying zedoary essential oil-induced apoptosis in NSCLC cells that are worthy of further study.