Grape pomace, a most abundant and renewable wine industry waste product was utilized as a suitable reducing, capping, and stabilizing biomolecules for green synthesis of silver nanoparticles (AgNPs). The physicochemical properties of biosynthesized grape pomace extract (GPE)-AgNPs were duly appraised via UV–Visible spectroscopy, X-ray diffractometer (XRD), Fourier-transform infrared spectroscopy (FTIR), and transmission electron microscopy. The analytical studies revealed that the GPE-AgNPs were well formed and stable in nature. The functional groups of organic molecules of GPE are present on the surface of AgNPs with average NPs diameter in the range of 20–35 nm. GPE-AgNPs exhibited significant free radical scavenging activity mainly DPPH radical (IC50, 50.0 ± 2.25 μg/mL) and ABTS radical (IC50, 38.46 ± 1.14 μg/mL). Additionally, the synthesized AgNPs showed noticeable inhibition of carbohydrate hydrolyzing enzymes mainly, α-amylase (IC50, 60.2 ± 2.15 μg/mL) and α-glucosidase (IC50, 62.5 ± 2.75 μg/mL). The GPE fabricated AgNPs showed noteworthy antibacterial potential against infectious bacteria viz., Escherichia coli and Staphylococcus aureus. The reaction mechanism of antibacterial activity was studied by measuring the bacterial cell membrane breakage and cytoplasmic contents, mainly, nucleic acid, proteins, and reducing sugar. Therefore, this research attempt illustrated the potential of GPE as a novel source intended for the biosynthesis of AgNPs that may open up new horizons in the field of nanomedicine.