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Staphylococcus aureus is competitive in milk and dairy environments; Pathogenic strains have been found to cause disease in their hosts throughout the world. Therefore, this study was designed to assess the prevalence of Staphylococcus aureus and determine their antibiotic susceptibility from lactating cow milk. A cross-sectional study was conducted in Bahir Dar dairy farms from October 2012 and March 2013.