Bovine mastitis is a costly disease in the dairy farms globally. The control of such disease is generally based on the prevention by the strict hygienic measures during milking. Other approaches include vaccination and the application of antibiotics. Regardless to these procedures, mastitis is not entirely under the control, thus increasing the need for alternative tactics. This study was conducted to isolate and identify lactic acid bacteria (LAB) from fresh cow's milk which possess antibacterial activity that could be used for mastitis control. 146 isolates were recognized as (LAB) from105 milk tanks samples after being cultured anaerobically on de Man, Rogosa and Sharpe (MRS) agar plates for 48 hours at 37 ^oC and identified by general bacteriological investigation. Afterwards, 24   isolates were identified to belong to genus Lactobacillus using polymerase chain reaction (PCR),  and for species level recognition MALDI-TOF MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) was used resulted in : L. fermentum (5), L. brevis (3), L. plantarum (4), L. paracasei (2), L. rhamnosus (3), L. pentosus (2), L. casei (3), L. raffinolactis (1) and L. mesenteroids (1). The antimicrobial activity of these strains against one of the major mastitis pathogens, S. aureus,was detected by the agar well diffusion assay and the  modified double layer method , where L. casei, L. fermentum andL. plantarum possess the most inhibiting effect besides they have no hemolytic nor gelatin liquefaction activity when their safety profiles were evaluated. The result of the antibiotic susceptibility test revealed that these isolates were resistant to vancomycin (VA), neomycin (N) and gentamycin (CN). On the other hands, they were highly sensitive to amoxicillin clavulanic acid (AMC), levamisole (LEV), tetracycline (TE) and penicillin (P).The study suggests that L. casei, L. fermentum and L. plantarum are perfect candidates to be used as probiotics to help in   preventing and controlling bovine mastitis caused by Staphylococcus aureus as they were proven to be safe and have antimicrobial activity againstthe organism.