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This study shows that rhamnolipid mixtures of MR01 and MASH1 have antimicrobial efficacy against Gram positive bacteria and may be evaluated as antimicrobial agent against multidrugresistant clinical pathogenic isolates. Background:In recent decades, biological properties of biosurfactants, particularly glycolipids, including antimicrobial, antifungal, and anti-cellular effects have been projected in many research studies. The unique advantages of these compounds in terms of production and environment made them important as antimicrobial agents. Objectives: This study aims to evaluate probable antibacterial and antifungal properties of MR01 and MASH1 biosurfactants against several specified microorganisms. Materials and Methods:In this article antibacterial activities of two rhamnolipid mixtures of MR01 and MASH1 were studied. MR01 and MASH1 are glycolipid-type biosurfactants that are being produced by two strains of Pseudomonas aeruginosa MR01 and MASH1, respectively. Antibacterial effects of two biosurfactants were assessed by disc diffusion test method and determination of minimum inhibitory concentration (MIC). Results: They exhibit not only excellent surface activity but also remarkable inhibitory effect against Gram-positive bacteria. According to results, although none of two biosurfactants showed significant effects on Gram negative bacteria growth inhibition, both assessing methods confirmed the growth inhibition of Gram-positive bacteria by MR01 and MASH1 biosurfactants. Conclusions: According to results of this study, MR01 and MASH1 biosurfactants had antimicrobial efficacy against Gram positive bacterial groups. This effect is comparable to antibiotics and therefore the future use of these biosurfactants as broad spectrum antibiotics is highly promising. These features of biosurfactants (BS) should broaden its applications in new advanced technologies. Future studies will be performed on characterization and isolation of biologically active fraction of the rhamnolipid biosurfactant produced by P. aeruginosa strains. This bioactive compound may be evaluated as a potent antimicrobial agent to be applied against a panel of pathogenic micro-organisms including a few multidrug-resistant (MDR) clinical pathogenic isolates such as methicillin-resistant Staphylococcus aureus (MRSA) and other MDR pathogenic strains.