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Objectives: Remarkable potential of silver nanoparticles (NPs) makes its use efficient in both biological as well as industrial applications. Post harvested NPs, free from other hazards chemicals will be ideal for biological applications. Thus, plant mediated biological method is much focused than the available approaches. Methods: The present work focusses on fabrication of silver oxide NPs (Ag2ONPs) using methanolic bark extract of Diospyros montana and characterized through sophisticated analytical techniques. Results: UV–Vis indicated Ag2ONPs formation, FTIR analysis identified the functional groups, XRD investigation depicted the face centered cubic crystalline nature, XPS study determined the chemical state, EDX confirmed the purity, spherical shaped and ∼ 10–50 nm size, was evidenced by SEM and TEM respectively. Zeta potential denoted the stability of Ag2ONPs. Photocatalytic degradation of methylene blue was observed. Ag2ONPs showed significant anticancer effect against hepatocellular carcinoma cells (Hep G2), which is mediated through increased DNA damage, & autophagy and decreased mitochondrial membrane potential. The synthesized Ag2ONPs revealed significant zone of inhibition in gram-negative (Escherichia coli − 16.33 ± 2.57 and Pseudomonas aeruginosa-18.56 ± 1.57) and positive (Bacillus subtilis-22.26 ± 4.47, Staphylococcus aureus 18.65 ± 3.15) bacteria at 40 µg/mL of Ag2ONPs exhibiting its antibacterial property. Conclusions: This enlightens the synthesis of pure and stable Ag2ONPs by green synthesis unwrapping their pharmacological properties which may play a vital role in nanomedicine anchoring its therapeutic efficiency.