Aim: To evaluation the effect of Lactobacillus acidophilus on Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 with detection of some virulence factors. Methods: Two hundred and fifty specimens (stool) from children under five years for both sexes were collected from some hospitals. All isolates were diagnosed according to morphological characteristics, biochemical tests. Monoplex pattern of PCR was used also for detection different genes in (7) Escherichia coli )O157:H7 (isolates; include 16SrRNA, eae, lifA, Stx1,Stx2 that encoded for ribosomal RNA, intimin, lymphocyte inhibitory factor, shiga toxins. Three types of probiotics strains were obtained, Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus acidophilus (ATCC4356). Minimum inhibitory concentration (MIC) of cell free supernatants of Lactobacillus acidophilus was determined by employing different dilutions (1/2, 1/4, 1/8, 1/16, 1/32), to detect the concentration of probiotic that will inhibit E.coli (O157:H7) growth. Results: Results showed, 210 (84%) samples were identified as E.coli from 250 samples. The result showed (7) isolates were identified as Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 and showed all isolates of O157:H7 were positive for 16SrRNA gene with (213bp) and eae with (741bp), lifA with (712bp), only Stx1 gene appeared in all isolates with (446bp) and no bands with Stx2. Current result showed only cell free culture supernatant of Lactobacillus acidophilus has inhibitory activity against all E.coli (O157:H7) isolates with different dilutions (1/2, 1/4, 1/8, 1/16, 1/32), while Lactobacillus fermentum, Lactobacillus plantarum have no effect against E.coli (O157:H7). The result showed the bacteriocin has inhibitory effect against E.coli (O157:H7), while organic acids and hydrogen peroxide haven’t any role in inhibition. The (MIC) value was (1/8) which inhibits the bacterial growth of isolates.