Abstract: Background and Objectives: Several infectious diseases became a big threat worldwide after spreading of multidrug resistant pathogens. Rayab milk found to be rich source of lactic acid bacteria with potential probiotics activity. Objective of the study was to evaluate the probiotic activity of LAB strains isolated from commercially fermented Rayab milk and to compare API identification method for some LAB isolates versus 16S rDNA sequencing molecular method. Materials and Methods: Lactobacillus MRS agar and broth media were applied to selectively isolate LAB from 20 fermented Rayab milk samples. API systems were used for biochemical identification. 16S rDNA sequencing had been used to identify ten randomly selected LAB isolates. Probiotic properties of all isolated LAB were tested, acid tolerability at pH 2.0, 3.0 and ability to grow in the presence of 0.3% bile salts, inhibitory effect of 10 antibiotics and bacteriostasis effect on food pathogens. Results: Ninety-five LAB had been isolated from 20 Rayab milk samples, 45 isolates out of them had been identified using API systems and grouped into four main genera previous reports, 62.2% of 45 isolates (28 isolates) showed distinguish resistance for surviving on low pH conditions. Fifteen out of these 28 isolates showed a tolerance to 0.3% bile salt. Moreover, 38 (84.4%) LAB isolates exhibited inhibitory activity against Staphylococcus aureus strain, Listeria monocytogenes strain (A), Salmonella typhimurium strain and E. coli O157 strain. Highest inhibition influences against LAB were observed for gentamicin, tobramycin and neomycin. While the lowest inhibition influences were recorded for amoxicillin, ceftriaxone, cefoxitin and nalidixic acid. Conclusion: LAB isolates with potential probiotic activity can be used to promote health and to combat food borne diseases.