Six bacterial isolates (four of them belonging to Pseudomonas aeruginosa and the other two were belonged to Pseudomonas spp.) isolated previously, were identified and/or ensured their identification. Results showed that these isolates were belonged to P. aeruginosa according to morphological, physiological, and biochemical characteristics, which was confirmed by using API 20E system. All these isolates were screened for their ability to produce rhamnolipid biosurfactant depending on surface tension measurment (mN/m), which referred that all these isolates were capable of producing rhamnolipid, and the best one was P. aeruginosa RB67. In order to get rhamnolipid hyperproducer mutants, mutagenesis of P. aeruginosa RB67 using UV light and MNNG were performed. The lethal and mutagenic effect of UV light and MNNG were studied on this bacterium. Results showed that this bacterium was sensitive to UV and MNNG. Fifty colonies from each treatment (UV and MNNG) were selected and screened for their ability to produce rhamnolipid semiquantitavely by replica plated on blood agar. Results indicated that all colonies showed approximately the same hemolytic zone. So, these colonies were replica plated on CTAB-methylene blue agar (Sigmend-Wagner medium) as an alternative method. Depending on this method twelve colonies from each treatment (UV and MNNG) were selected (based on the formation of largest dark blue halo against light blue background) and used for measuring rhamnose concentration. Results showed that these mutants were varied in their ability to produce rhamnolipid. Two mutants from each treatment showed an increase in rhamnolipid production and the highest rhamnose concentration (94 ?g/ml) was gained by the mutant (MOM12) compared with rhamnose concentration (70 ?g/ml) produced from P. aeruginosa RB 67 (wild type). In order to qualify the rhamnolipid produced from P. aeruginosa RB67 (wild type), UOM12, and MOM12, Fourier transformer infrared (FTIR) spectroscopy was performed. Results showed that there are no apparent qualitative differences in rhamnolipid produced from these three isolates.