Background: With growing environmental concern, interest was increased in research anddevelopment of environmentally friendly substances. Microbial surfactants are nontoxic andbiodegradable bioproducts that are widely used in medical, pharmaceutical, agricultural,industrial, food, cosmetics, and refinery applications. However, the main challenges in theproduction of biosurfactants are the high production cost and the limited yield. The improvementof biosurfactant production depend on many strategies, such as the use of low-cost raw materials,adjusting medium and growth conditions, and the selection of hype-producing strains.Objective: The study was conducted to obtain Pseudomonas guguanensis strain Iraqi ZG.K.Mmutant with higher biosurfactant production, through random mutagenesis.Materials and Methods: Chemical mutagen (acridine orange) in different concentrations andthe physical mutagen (UV irradiation) were used for the production of random mutagenesis inPseudomonas guguanensis strain Iraqi ZG.K.M. The biosurfactant hype-producing strain wasselected depending on CTAB test, E 24 %, and the measurement of surface tension.Results: A clear indication of the efficiency of UV light in producing biosurfactant mutant. Inwhich, a higher E 24 % (78%) and the lowest surface tension (33 mN/m) were recorded withZUVM2 mutant.Conclusion : UV light can successfully used to mutagenize Pseudomonas guguanensis strainIraqi ZG.K.M to obtain hyper-producer mutants of biosurfactant that used in many applications.