Background: Probiotics are health promoting microorganisms incorporated into various kinds of food. Members of Lactobacillus and Bifidobacterium are the most commonly used. Objectives: This study aimed to isolate Lactobacillus and Bifidobacterium from different products at Assiut Governorate, to detect its antibacterial activities against pathogenic bacteria, to evaluate probiotic, haemolytic and enzymatic activates, to compare between conventional methods for identification and PCR as gold standard. Methodology: Isolation and identification of Lactobacillus and Bifidobacterium by conventional methods. Probiotic properties were determined by growth at different temperatures, different NaCl concentrations, pH and bile tolerance. Antibacterial activity examined against three indicator pathogenic organisms. PCR was used for identification of Lactobacillus and Bifidobacterium species. Also haemolytic and enzymatic activities were determined. Result : The identified Lactobacillus spp. were L. acidophilus 54(29.5%), L. fermentum 50(27.3%), L. rhamnosus 34 (18.6%), L. plantarum 27(14.8%), L . paracasei 13(7.1%) and L.GG 5(2.7%) .The Bifidobacterium species were B. Breve 59(31.7%), B. dentium 42 (22.6 %) , B . bifidum 53 (28.5%) , B . subtile 15(8%) B. longum 6(3.2%), B . animalis 7 (3.7% ) and B. infantis 4 (2.2%). All the isolates tolerated low pH and bile salts, non heamolytic and had good antibacterial activity. The sensitivity and specificity of the conventional methods was (94.7%, 95.97%) and (93.86%, 94.97%) in Lactobacillus and Bifidobacterium respectively. Isolated strains produced β-galactosidase which is beneficial for lactose intolerance and did not produce carcinogenic enzymes. Conclusion: Rayeb, yogurt, milk powder and cereals can be used as potential source of probiotics because they tolerate acidic media, bile salts, have good antibacterial activity. Also, they have beneficial enzymatic activities. PCR was a rapid method for identification of probiotic strains.