To investigate the effect of different protocols for estrus synchronization in ewes on the vaginal tract bacteria and evaluate the sensitivity of bacteria to different antibiotics. Sixteen local breed ewes were allocated to four groups, each containing four. Group A: ewes treated with sponges for 10 days; Group B ewes treated with CIDR for 10 days; Group C ewes treated with sponges for 14 days and finally Group D ewes treated with CIDR for 14 days. Sterile swaps were taken from the vaginaʼs mucous before insertion of sponge or CIDR and after removing it for each group. Vaginal swabs from synchronized ewes were obtained from the vaginaʼs mucous before insertion of sponge or CIDR and after removing it for each group to use in different bacteriological tests. The results showed that the most dominant bacteria were E-Coli 7/7 (100%), and Staphylococcus aureus (71%) before insertion, while in group A, the percentage was 75% and 100% for E-Coli only in group B and group C respectively. According to E-coli, the results showed that it was more sensitive to Doxycycline (3.36±0.3), Enrofloxacin (3.23±0.2), and Ciprofloxacin (3.18±0.3) figure (2). Also, Staphylococcus aureus was sensitive to Doxycycline (3.32±0.4), Enrofloxacin (3.17±0.3), and Ciprofloxacin (3.12±0.3). It was concluded that the E-coli was the most prominent microflora in the vagina in ewes before and after insertion of sponges. Doxycycline, Enrofloxacin, and Ciprofloxacin are the most effective antibiotics against E-coli and Staphylococcus aureus.