Vitis viniferaL., commonly known as grape is a major fruit crop in the world. Grapes seem to confer health benefits due to their chemical components, biological and antioxidant activities. The present study is conducted to evaluate the biochemical constituents, antioxidant, and antimicrobial potential of ethanolic grape peduncles (EGP) extract. The result of phytochemical analysis revealed the presence of various phytochemicals such as flavonoid, tannin, carbohydrates, alkaloids, cardiac glycoside, phenol, steroid, terpenoids, quinones and anthraquinones. Furthermore, total phenolic content (TPC) and total flavonoid contents (TFC) were 7.35 ± 0.25 mg GAE/g (Gallic Acid Equivalent per gram) and 29.67 ± 0.13 mg QE/g (Quercetin Equivalent per gram) respectively. DPPH (2, 2-diphenyl-1-picrylhydrazyl) free radical scavenging assay revealed IC50= 159.3 μg/mL. The antibacterial and antifungal study disclosed that the extract was highly potent againstSalmonella typhiwith maximum zone of inhibition of 27.2 ± 1.60 mm andEpidermophyton floccosumwith 74 ± 1.81% inhibition. The extract was analyzed for its cytotoxicity and antileishmanial activity and showed no activity against HeLa cell line and promastigotes ofLeishmania major. Elements Fe, Mn, Ni, Pb and Cd were determined by atomic absorption spectroscopy and approximately 50 compounds were identified by Gas Chromatography-Mass Spectrometry (GC–MS). Current work suggest that grape peduncles can be a promising source of bioactive medicinal component.