This study aimed to evaluate the most predominant strain of avian pathogenic Escherichia coli (APEC) that causes colibacillosis with respiratory manifestation in broiler chickens. Isolation and serotyping identification for the isolates of E. coli were performed. Prevalence of APEC differentiated by detection of the 8 virulence-associated genes “astA, iss, irp2, papC iucD, tsh, vat, and cva/cvi" using multiplex PCR technique in the most important isolates of E. coli was also investigated. Antibacterial sensitivity test for antibiotics disc and oregano at different concentrations (20%, 15%, 10%, and 5%), were estimated for the most important isolates of E. coli. A total of 50 broiler farms were located in Gharbia, Giza, Menoufia, Bahaira, and Dakahliah Governorates were examined. Specimens from lung, liver, spleen, and kidney from diseased birds were used for bacteriological examination. Results revealed that E. coli O27 was the most predominant serotype strain (11 out of 35 isolates), followed by the serotypes of O146 (4/35), O Untypable serotypes (4/35), and the (1/35) of each serotype of O8, O20, O44, O78, O114, O125, O142, O152, O153, O158, O164, and O166. One isolate of APEC O27 out of 11 isolates has 6 virulence-related genes “astA, iss, papC, iucD, vat, and cva/cvi". Difloxacin and oregano at a concentration of 20% have the highest percentage (100%) of sensitivity against 11 isolates of E. coli O27 strains. In conclusion, E. coli O27 was the most predominant serotype infecting farms of broiler chickens. Difloxacin and oregano 20% could be used for the treatment of APEC O27.