Were collected 154 milk samples from 120 animals included (25 sheep, 25 goats, 70 cows), subjected these samples to California Mastitis Test,(60) sample showed a positive result. These samples were grown at different common, selective culture media, and depended on the study of the cultural characteristic and microscopic examination and biochemical tests, 32 isolates diagnosed as Staphylococcus aureus, 11 isolates diagnosed as Staphylococci coagulase negative and 17 isolate as gram negative bacteria, the diagnosis was confirmed by using the API STAPH system. The staphylococcus aureus isolates were tested for their sensitivity to commonly used antibiotics. the isolates are more susceptible to Chloramphenicol, Streptomycin and Ciprofloxacin, and the least sensitivity was observed to Ampicillin, Gentamycin and Cefixime. then selected one isolate from S. aureus (isolate exhibit resistant to large number of antibiotic), for determination antibacterial activity of Punica granatum in different concentration 25mg/mL, 50mgL/mL, 75mgLmL, 100mg/mL150, mgL/mL, the mean of inhibitory zone diameter ware 20.66, 23, 24.66, 27, 29.66 respectively The present study provides evidence antibacterial effect of aqueous extract of Punica granatum on resistant staphylococcus aureus isolated from mastitic milk.