In this research, a study was carried out to assess the antagonistic ability of Lactobacillus obtained from raw milk and various dairy products “Karish cheese, fermented milk, yogurt, and ice cream". From a total 100 samples that were examined, 74 of them comprised lactic acid bacteria whereby 36 of them were related to genus Lactobacillus. The isolated strains examined to their ability to form biofilm, and most isolates showed medium to strong biofilm formation. All isolated Lactobacillus strains exhibited the ability to inhibit biofilm formation by pathogenic bacteria, including Escherichia coli, Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes. The in-vitro antimicrobial activity of the Lactobacillus isolates was evaluated against several pathogenic strains. The results showed that all examined isolates had a strong antibacterial effect against Staphylococcus aureus, with inhibition zones ranging from 10 to 17 mm. The antimicrobial activity of extracted bacteriocins from the Lactobacillus isolates differ greatly. The bacteriocins from six isolates of lactobacillus showed antibacterial pursuit against the tested pathogens, with inhibition zones ranging from 1 to 7 mm. In contrast, the bacteriocins from four isolates exhibited low activity against the tested pathogens. Molecular identification for the isolated strains (n=5) using 16S rRNA gene sequencing revealed that three isolates were related to Limosilactobacillus fermentum that registered in Gene-bank with accession numbers (PP784302- PP784303- PP784304), while the other two isolates were related to Lactiplantibacillus plantarum and registered with accession numbers (PP788561- PP788562).