The present study focused on identifying antimicrobial and antioxidant bioactive fraction from Syzygium cumini leaf extract. Initial screening through contact-thin layer chromatography (TLC) bioautography identified active regions, which were subsequently subjected to partial purification by preparative TLC (PTLC). This partially purified fraction was characterized using UV-Visible (UV-Vis) Spectroscopy, ATR-FTIR, and GC-MS. Antimicrobial activity against Staphylococcus aureus and Mucor species was evaluated using the disc diffusion assay, while antioxidant potential was assessed through the DPPH radical scavenging assay. The solvent system containing ethyl acetate, acetic acid, formic acid, and water (7:1:3:2.5) successfully separated a distinct chromatographic spot exhibiting antimicrobial activity. UV-Vis analysis exhibited a λ_max peak at 224 nm. ATR-FTIR spectra revealed the presence of functional groups, including O-H (phenols), C=C (aromatic rings), and C=C (alkynes), suggesting a phenolic nature of the compounds. GC-MS analysis identified 17 compounds within this bioactive fraction. Notably, this purified fraction demonstrated significant antimicrobial properties, with inhibition zones of 21.77 ± 0.68 mm against S. aureus and 24.93 ± 0.80 mm against Mucor sp., and also possessed significant antioxidant activity (IC₅₀ value of 56.75 µg/mL).