This project was conducted to evaluate the activity of S-layer proteins isolated from Lactobacillus in comparison with the activity of concentrated filtrate of Lactobacillus against some pathogenic microorganisms and against tumor cell lines in vitro. Twelve isolates of Lactobacillus spp. obtained from, vinegar, human milk, cow milk, yoghurt and vagina, were used to detect the S-layer protein (Slp) by Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis (SDSPAGE) then extracted it by excised the Slp pand and treated with 6M guanidin hydrochloride (G-HCl) to eluted the protein from gel. The Molecular weights (MW) of Slps were estimated between (37-63 kDa) depending on the Lactobacillus species. The concentrations of Slp were estimated by using a Kit based on the Biuret method. One isolate of each of Lactobacillus acidophilus and Lactobacillus casei, were selected depending on the MW and concentrations of S-layer proteins. The inhibitory effect of Lb. acidophilus and Lb. casei was determined against pathogenic microorganism; Pseudomonas aeruginosa , Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, and Candida albicans on solid and liquid MRS media. Results revealed that less inhibitory activity against tested microorganisms was detected on the solid medium, compared to the liquid one. Greatest inhibitory effect of L. acidophilus and L. casei was appeared against P. aeruginosa when the zone of inhibition reached to 24 and 22 mm, respectively. It appeared that the inhibitory effect of L. acidophilus was more than that of L. casei against most of tested microorganisms, while S-layer proteins have no effect against pathogenic microorganisms.