In Iraq, sheep play an important role in supplying the local market’s requirements for milk and meat. The objective of this study, 117 milk samples collected from Awassi ewes put to sale in the animal sales yard in Al-Qasim District from Babylon Governorate during four weeks on December 2023. The udder and milk inspection by visual examination and indicator papers (BOVIVET) for detection of mastitis. Consequently, cultured mastitis milk sample on HiCrome™ Klebsiella Selective Agar Base for isolation and easy detection of Klebsiella spp and phenotypic detection of Escherichia coli (EC) on MacConkey agar. Consequently, Staphylococcus aureus is isolation by inoculating on Mannitol salt Agar, and confirmed by the Vitek2 system, then antibiotic resistance characteristics are studied by the Kirby-Bauer technique on Muller Hinton agar. The results showed phenotypic detection of 24(20.5%) mastitis milk sample out 117 milk samples and isolation of Klebsiella spp 5(20.8), Escherichia coli 7(29.1) and Staphylococcus aureus 12 (50%) then studying the characteristics of microbial resistance to Staphylococcus aureus isolates , it was shown that it possesses resistance to Ciprofloxacin 3(25%), Ampicillin 7(58.3%), Gentamycin 3(25%), Streptomycin 2 (16.6%), Amikacin 3(25%), Doxycycline 5(41.6%), Cefaxitin 2(16.6%), Ceftazidime 2(16.6%), Ceftazidime 2(16.6%) and Erthromycin 2(16.6%). In conclusion, data showed there are many causes of mastitis in sheep, and Staphylococcus aureus bacteria is still the most common cause among other causes, and the most important thing is that it acquires resistance due to mismanagement and indiscriminate use of veterinary medicines. Therefore, we recommend periodic follow-up of sheep, giving necessary vaccines, and educating farmers to limit the spread of diseases that effects animal and human health and cause significant economic losses.