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Study design of cases is a cross-sectional study that descriptive study design of 280 isolates collected from tonsillitis, the identification achieved by using Vitek2-GP.The biological activity of antimicrobial sensitivity test of all 280 isolates against antibiotics including Ceftazidine 70(25%)resistance, 196(70%) sensitive, 14(5%) intermediate; Azithromycin 154(55%) sensitive, 28(10%) intermediate., 98(35%) resistance; Oxacillin 266(95%) sensitive, 14(5%) resistance; Cephalexin 238(85%) sensitive, 42(15%) resistance; Gentamycin 266(95%) sensitive, 14(5%) resistance; Ceftriaxone 238(85%) sensitive, 42(15%) resistance;Norfloxacin 252(90%) sensitive, 28(10%) resistance; Cefoxitin 266(95%) sensitive, 14(5%) resistance; Erythromycin 42(15%)sensitive, 238(85)% resistance; Cloxacillin 126(45%) resistance, 154(55%) sensitive; Imipenem 266(95%) resistance, 14(5%) sensitive; Vancomycin 266(95%) resistance, 14(5%) sensitive and Methicillin 280(100%) resistance, 0 (100%) sensitive) compared with CLSI(2013).Radial caseinolysis assay and Fibrinolysis assay of Staphylococcal fibrinlysin achieved by quantitative screening of staphylokinase on plasma agar plate and semi-quantitative screening of staphylokinase on skimmed milk agar plate to limit fibrinolysis zone and caseinolysis zone around well on plat. Results showed variable degree of production staphylokinase, some isolates positive for production, another negative for production staphylokinase (staphylococcal fibrinolysin).Induced mutation achieved by chemical mutagenesis when exposition S.aureus to different mutage of Acridine Orange (AO) chemical mutagen in dose (10,20,30,40,50,60) mg, the results of mutagenesis in optimum mutage (dose of mutagen) with increase production of staphylococcal fibrinolysin when occurs random mutagenesis was 75 colony (viable cells) viability in 30 mg.Genotypic analysis of sak gene encoded for staphylokinase accomplished by using PCR, all results of S.aureus isolated from tonsillitis positive for possessing sak gene (400bp).  How to cite this article: Mohammed, N.R. and AL-Auadi AL-Musawi, S.J. (2019). Mutagenicity Acridine Orange Mutagen On The Biological Activity of S.aureus Isolated From Tonsillitis. International Journal of Drug Delivery Technology, 9(4): 693-696. Source of support: Nil. Conflict of interest: None.