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The synthesis of nanomaterials using plant extracts is considered to be a safer, environmentally friendly, and cost‐effective alternative method. In this work, Eysenhardtia polystachya (EP) extract and choline chloride/urea (ChCl/urea)‐based deep eutectic solvent (DES) were used for the synthesis of gold nanoparticles (AuNPs), without an additional stabilizing agent, which is of great interest in the field of biomedicine. The antioxidant capacity of the extract and the phenolic content, involved in the reduction of Au3+ ions of the precursor were evaluated. The AuNPs were characterized by ultraviolet‐visible spectroscopy, Fourier transform infrared spectroscopy, X‐ray diffraction, dynamic light scattering, ζ‐potential, confocal laser scanning microscopy, scanning and transmission electron microscopy. The nanoparticles are mostly quasi‐spherical shape, with a multiply‐twinned structure, an average particle size of 36 nm, and a characteristic absorption peak at 545 nm. The colloidal stability was confirmed with a ζ‐potential value of −32.8 mV. Finally, the AuNPs were tested for their potential in cancer therapy using human prostate adenocarcinoma PC‐3 cells. The anti‐inflammatory activity of the EP extract and AuNPs was also evaluated. The results showed that the synthesized AuNPs could potentially induce cell death and have an anti‐inflammatory effect. [ABSTRACT FROM AUTHOR]