To characterize the phytoconstituents and explore the antibacterial efficacy, cytotoxicity of Moringa oleifera leaf extract, assessing its potential as natural therapeutic alternative for antimicrobial and anticancer applications. Moringa oleifera leaves were collected, air-dried, and extracted using 50% aqueous methanol. Fractionation was carried out using solvents of increasing polarity and concentrated for analysis. The method of disc diffusion was utilized to evaluate the antimicrobial effects against Escherichia coli, Shigella boydii, and Staphylococcus aureus, with standard antibiotics as controls. Cytotoxicity was assessed via the MTT assay on A-549 (human lung adenocarcinoma cell lines) and BEAS-2B (non-tumorigenic human bronchial epithelium cell lines). Spectroscopic characterization (UV-Vis and IR) was conducted to determine the bioactive compounds responsible for the observed biological activities. The Moringa oleifera methanolic extract exhibited a strong antibacterial effect against bacteria such as E. coli and S. aureus, increasing the diameter of the zone based on their dose. However, Shigella boydii showed resistance. Cytotoxicity analysis revealed selective toxicity, with up to 70% cell death in A-549 cancer cells at 50 µg/ml, while normal BEAS-2B cells exhibited 38% cytotoxicity at the same concentration. Spectroscopic analysis confirmed the presence of flavonoids, phenolic acids, carotenoids, and chlorophyll derivatives, known for their antimicrobial, anticancer, and antioxidant properties. The calculated band gap (3.34 eV) indicated strong UV absorption, which may contribute to reactive oxygen species mediated antimicrobial and anticancer mechanisms. Moringa oleifera leaf extract demonstrated potent antibacterial activity and selective cytotoxicity against cancer cells while exhibited minimal toxicity to normal cells. Its rich phytochemical profile suggests promising role as natural antimicrobial and anticancer agent.