We previously reported that MR01, an indigenous strain of Pseudomonas aeruginosa, was able to produce a rhamnolipid-type biosurfactant. Here, we attempted to define the structural properties of this natural product. The analysis of the extracted biosurfactant by thin-layer chromatography (TLC) revealed the presence of two compounds corresponding to those of authentic mono- and di-rhamnolipid. The identity of two structurally distinguished rhamnolipids was confirmed by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Liquid chromatography/mass spectrometry (LC/MS) of extracted biosurfactant revealed up to seventeen different rhamnolipid congeners. Further quantification showed di-rhamnolipids as the major compound (77.2%), while monorhamnolipids comprising a smaller proportion (22.8%) of MR01 biosurfactant. Rha-Rha-C10-C10 was verified as the major component of the MR01 biosurfactant (35.93%). Cytotoxic activity of MR01 biosurfactant against human cancer Hela cells showed an excellent inhibitory effect of 5μg/ml. An isolated mutant strain (MR01-C) created by Gamma ray irradiation demonstrated more than one and a half-fold biosurfactant production and activity compared with the parent strain. Analysis of the biosurfactant produced by MR01-C showed the magnitude of di-rhamnolipids in the sample increased up to 88.6% (∼15% higher than control) and the quantity of Rha-Rha-C10-C10 increased to 52.08% (∼45% higher than control).