Bacterial infections and cancer has been the most prevalent diseases throughout the World. The discovery and development of new antibacterial and anticancer agents are critical. If such a valuable medicinal product is created from waste resources from the oil refinery business, academicians and industrialists will pay close attention. The sequential extraction process followed by RP-HPLC fractions was used to purify and identify a new peptide from mustard oil refinery sludge. The sequence of the peptide was determined as NH2-KYQFFVP-COOH using MALDI TOF/TOF Mass spectrometry. The antibacterial activity of the peptide was determined against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus subtilis. The proapoptotic activity of the peptide was also determined against Hep2 (Human epidermoid cancer cell line). These findings suggest that readily available bio-waste might be a novel source of bioactive antibacterial and proapoptotic lipopeptides.