Aim: The aim of the present study was to evaluate immune modulatory effect of fractions of D. stramonium L. leaves on human peripheral blood mononuclear cells (PBMC) followed by assessment of cytotoxic abilities of immunomodulated PBMC toward cancer cells. Material and methods: Bioassay (PBMC proliferation) guided fractionation of methanolic leaf extract of D. stramonium was performed to get active fraction and LC-MS was performed to identify the phytocompounds present in the bioactive fraction. The immunomodulatory potential of D. stramonium active fraction was assessed by i) MTT microcytotoxicity assay using A549 (lung carcinomas) and MCF-7 (breast cancer) cell lines and ii) analyzing the production of IL-2 and IFN-γ by human PBMC in the presence of active fraction. Results: Chromatographic fractionation guided by PBMC proliferation assay of D. stramonium extract resulted in bioactive fraction (fraction-10) exhibiting significant immunostimulatory activity [EC50=19.1±1.5 (μg/ml)] on human blood lymphocytes. Fraction-10 pretreated PBMC displayed enhanced cytotoxicity towards A549 and MCF-7 (59%±2.1% and 62%±2.3% at 1:20 effector:target ratio respectively). Moreover, fraction-10 also enhanced the secretion of IL-2 (8 fold) and IFN-γ (10 fold) by human PBMC. The preliminary phytochemical analysis of fraction-10 from D. stramonium showed the presence of terpenoids and steroids. LC-MS analysis depicted presence of four major phytoconstituents in fraction-10 as daturaolone, daturadiol, stigmasterol and sitosterol with corresponding mass spectrum (m/z) of 440, 442, 412 and 414 respectively. Conclusion: The present report concluded that active fraction-10 of D. stramonium possesses potential immunostimulators that are capable of enhancing anticancer responses of human blood lymphocytes.