Background: Atropa acuminata plant, which is also known as the maitbrand or Indian belladonna, belongs to the family Solanaceae and is closely related to the deadly nightshade of Europe and North Africa used in the treatment of various diseases. Objectives: The main objective is to investigate the antidiabetic potential of silver nanoparticles of Atropa acuminata. Methods: In this study green synthesis of silver nanoparticles was carried out. The current study engross the green synthesis of A. acuminata roots by reducing silver ions, where ultraviolet (UV) spectral analysis and transmission electron microscopy confirmed nanoarchitecture. The optimized silver nanoparticles were characterized for shape, size and morphological features by various techniques viz., SEM, TEM EDXA and XRD. The optimized formulation was further subjected for lipid peroxidation assay and in vitro antidiabetic assay in order to understand the antidiabetic potential of formulated silver nanoparticles. Results: The Silver nanoparticles of Atropa acuminata roots was successfully prepared by optimizing different concentration of plant extract at different temperatures and stirring speed. The Resultant optimized nanoparticles showed a particle size around 20 nm and -28 mv zeta potential. Further the characterization of optimized AgNPs was carried out. SEM provides the shape, size, and morphological features, whereas EDXA confirms the compositions and distribution of nanoparticles through spectrum and elemental mapping. XRD diffraction analysis revealed the crystalline structure of nanoparticles. Further, Nanoparticles showed a maximum scavenging potential of 82.633±0.116 for superoxide anion free radicals at 100 µg/mL concentration. Among two anti- diabetic assays, the αamylase assay shows a better result of percent inhibition 63 ± 1.32 at 75 μg/ml concentrations. Lastly, in vitro, drug release study revealed a 101.50% cumulative release from Ag- NPs formulation up to 1 hour, which was better than the standard one. Conclusion: This study explores the novel technology of green synthesis for various biomedical applications.