Clinical mastitis is a common disease found in dairy ewes worldwide that results in great economic losses. This study was designed to investigate the prevalence rate of Staphylococcus aureus and Streptococcus agalactiae isolated from clinically mastitic ewes, determine the virulence determinant genes, and identify antimicrobial sensitivity assay of the isolated bacteria. A total of 37 milk samples were collected from 27 ewes diagnosed with clinical mastitis. These revealed 12 isolates of S. aureus (32.4%), followed by 7 isolates of S. agalactiae (18.9%). Real-time polymerase chain reaction was used to identify eight strains of S. aureus as possessing the thermonuclease (nuc) gene, and five isolates of S. agalactiae were positive for the presence of the cell surface protein (cfb) gene. Eight isolates of S. aureus and five isolates of S. agalactiae isolated from dairy ewes with clinical mastitis in El-Sharkia, Egypt, possessed several virulence determinants. The antibiotic sensitivity pattern was performed and a large proportion of isolates of S. aureus were found to be sensitive to Vancomycin, Kanamycin, Ciprofloxacin, and Gentamycin, whereas large proportions were highly resistant to Penicillin G and Tetracycline. S. agalactiae isolates revealed full sensitivity to Vancomycin, Ciprofloxacin, and Oxacillin, but they were highly resistant to Kanamycin and Penicillin G. The virulence gene should be considered when developing prevention and treatment program for clinical mastitis in ewes in regional areas of isolation. It is necessary that the antibiogram findings of drugs with the highest sensitivity be used to replace the currently used antibiotics with those that are more effective.