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The current study deals with the investigation of the antioxidant, anti-inflammatory and immunomodulatory properties of the essential oil from Datura stramonium leaves (D. oil). The GC-MS analysis showed that the dominant compounds present in the D. oil were neophytadiene (Phytol acetate) (10.76%), β-damascenone (9.67%), and β- eudesmol (7.2%). D. oil exhibited in vitro scavenging potential of free radicals by DPPH and ABTS assays (IC50 values 71.35 ±1.06 μg/ml and 61.01 ± 1.07 μg/ml, respectively). We found that D. oil decreased the nitric oxide production in LPS-stimulated J774A.1 cells by 52.43% without affecting their cell viability. D. oil was found to stimulate the proliferation of human peripheral blood mononuclear cells (PBMC) and, also enhanced the secretion of IL-2, IFN-γ and TNF-α. Furthermore, D. oil treatment of PBMC induced the expression of CD3, CD8, and CD56 and intracellular granulysin levels in the immune cells. The treatment of human lymphocytes by D. oil enhanced their ability to kill colon cancer cells HCT-116 (51.09 ± 7.5%) and SW620 (48.57 ± 8.08%) at 20:1 (effector: target ratio). Moreover, these activated lymphocytes cause target cell death by reactive oxygen species and by damaging mitochondrial membrane potential of these cells. Taken together, the current findings showed D. oil as immunotherapeutic agent which can be used for colon cancer treatment.