This study aims to assess the potential anticancer activity of lemongrass essential oil (LEO) using in vitro and in silico methods. The steam hydrodistillation of the aerial parts yielded 3.2% (wt) LEO. The GC-MS analysis of the LEO revealed the presence of α-citral (37.44%), β-citral (36.06%), linalool acetate (9.82%), and d-limonene (7.05%) as major components, accompanied by several other minor compounds. The antioxidant activity, assessed using the DPPH assay, revealed that LEO exhibits an IC value of 92.30 μg/mL. The cytotoxic effect of LEO, as well as LEO solubilized with Tween-20 (LEO-Tw) and PEG-400 (LEO-PEG), against a series of cancer cell lines (A375, RPMI-7951, MCF-7, and HT-29) was assessed using the Alamar Blue assay; the results revealed a high cytotoxic effect against all cell lines used in this study. Moreover, neither one of the tested concentrations of LEO, LEO-PG, or LEO-TW significantly affected the viability of healthy HaCaT cells, thus showing promising selectivity characteristics. Furthermore, LEO, LEO-PG, and LEO-TW increased ROS production and decreased the mitochondrial membrane potential (MMP) in all cancer cell lines. Moreover, LEO treatment decreased all mitochondrial respiratory rates, thus suggesting its ability to induce impairment of mitochondrial function. Molecular docking studies revealed that LEO anticancer activity, among other mechanisms, could be attributed to PDK1 and PI3Kα, where the major contributors are among the minor components of the essential oil. The highest active theoretical inhibitor against both proteins was β-caryophyllene oxide.