Aim: This study intended to ascertain whether probiotics added to various food products exhibited antimicrobial and antibiofilm properties against common microbial pathogens. Materials and methods: Two hundred samples were collected (130 from Assiut and 70 from New Valley) from different sources including yogurt (90), rayeb (80), and milk powder (30). Lactobacilli strains were isolated and biochemically identified. Then genotypic characterization of the samples was done. The lactobacilli CFS's (CFS) antimicrobial activity was measured by agar well diffusion. The ability of these pathogens to form biofilms was tested. Furthermore, the microplate titer method was employed to analyze the antibiofilm characteristics of CFS. Result: Lactobacillus strains were isolated from different milk products and genotypically investigated and nineteen L. acidophilus have been isolated. Most concentration CFS show antibacterial activity against tested microorganisms with an inhibition zone of diameter greater than 25 mm. Moreover, The CFS of lactobacilli inhibited the biofilm formation by these pathogens.