The potent biosurfactant-producing marine actinomycete Micromonospora marina was isolated from the mucus of the scleractinia coral Acropora formosa. The strain was screened for biosurfactant production and showed positive signs for hemolytic activity, drop-collapse test, oil displacement, emulsification activity and lipase production. The yield of biosurfactant was found to be high when the medium containing sucrose and peptone were used as carbon and nitrogen sources, respectively. The chemical characterization of the biosurfactant confirmed the presence of proteins, lipids and carbohydrates, thus indicating that the biosurfactant was lipopeptide in nature. Further, the presence of palmitoleic acid in GC-MS analysis also confirmed that the produced biosurfactant was a surfactin derivative. The structural characterization of surfactin was confirmed by H-1 NMR and C-13 NMR, and the results showed the presence of mystric fatty acid (-CH3CH2-), palmitoleic acid, lipopeptide monoesters and aromatic protons and carbons. Further, the surfactin exhibited potential cytotoxic activity against human breast cancer cells by inducing apoptosis through the increase of ROS generation and cleaving mitochondrial membrane potential. Taken together, the surfactin produced by M. marina could act as an excellent anticancer agent against breast cancer.