Escherichia coli O157: H7 is an important pathogen that causes fatal infections. It has been the cause of several outbreaks in different areas world-wide and is associated with high mortality and morbidity. Multiple procedures were adopted to detect this serotype, but conventional culture techniques represent the gold standard. In this study we estimated peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) technique for the rapid detection of E. coli O157:H7. PNA-FISH technique was performed for 50 strains; 20 were E. coli O157 H7, 20 were E. coli O157 non-H7 strains, and 10 strains belonging to other Enterobacteriaceae. Sensitivity, specificity, and accuracy of the FISH technique were 100%, 86.67%, and 92% respectively with Confidence Interval of 95% (82.5% to 98.4%). Both PNA-FISH and conventional culture technique were used for examination of 100 non-heat-treated milk samples which were artificially contaminated with E. coli O157:H7. PNA-FISH sensitivity, specificity, and accuracy were 97%, 100%, and 98.5% respectively with a Confidence interval of 95% (95.7% to 99.7%). The PNA-FISH technique had approximate results to the conventional culture methods, in addition, it reduced the diagnosis time to one day.