P. aeruginosa M14808 was screened to produce rhamnolipid with high yield by mutation in a high magneto-gravitational environment. After silica gel columniation chromatogram, the components of the crude biosurfactant extract were analyzed by TLC, FTIR, and ESI-MS. The major components were characterized as rhamnolipids. The rhamnolipids consisted of two main types, mono- and di-rhamnolipids. Di-rhamnolipids showed significant anti-proliferative activity against human breast cancer cell line (MCF-7) at a minimum inhibitory concentration (MIC) of 1 μg/mL as opposed to a minimum inhibitory concentration of 5 μg/mL against human non-small lung cancer cell line (H460).