In recent years, a wide range of studies has pointed out the role of nanoparticles as reservoirs of therapeutics for several diseases, including cancer. Nowadays, cancer research is focused on the development of novel treatment approaches to fight this dreadful disorder. Based on the evidential research and applications of nanoparticles, it is expected that green synthesized nanoparticles may show a prominent role, especially in the biomedical field. The present work is centered on the preparation and characterization of silver nanoparticles (Ag-NPs) from the aqueous (AQ) extract and non-alkaloidal (NA) fraction of Datura stramonium leaves and to evaluate their anticancer potential against mammalian cell lines. The biogenic Ag-NPs are characterized by UV-vis spectra, FTIR DLS, UV-Vis, SEM, and TEM. SEM and TEM analysis reveals the spherical morphology of NPs. The Ag-NPs exhibit cytotoxicity against various mammalian cell lines (A549, HCT-116, PANC-1, SHSY5Y, and U87), which indicate that the AQ and NA based NPs are highly potent to cause cancer cell death. To the best of our knowledge, the present report, for the first time, describes the green synthesis of Ag-NPs from the NA fraction of the D. stramonium and provides pieces of evidence for its anticancer potential.