The main serotypes, pathogenicity, and antibiotic susceptibility of Avian Escherichia coli isolated from Colibacillosis cases in the Aljabel Al Akhdar region of Libya were investigated in this study. To determine the pathogenicity of E.coli isolates, Congo red binding, hemolysis, and lethality tests were conducted on Seventy-two unvaccinated broilers chicks on the day of hatching. Avian pathogen serotypes and antibacterial susceptibility testing to twelve antibiotics were also performed according to the standard methods. Results revealed that the only (O114) serotype that had been orally inoculated caused mortality on the first day. While all E.coli isolates inoculated by subcutaneous route showed mortality on the first day. The highly pathogenic serotypes were (O26) induced (100%) mortality. Congo red (CR) binding assay showed that (100%) of isolates were positive. Only (O26) was found to be hemolytic. Serotyping results revealed that E.Coli strains belong to serotypes (O26, O111 O114, O119). According to the results, (100%) of E.Coli isolates were less sensitive to Cephalothin, Lincomycin, as well as Erythromycin, while the sensitivity level to Amoxyclav and Doxycycline was (40%)and(60%) respectively. Only (20%) of E.Coli isolates showed resistance to Enrofloxacin, Neomycin, Streptomycin, and Tetracycline. All strains were susceptible to Imipenem (100%). Significantly, O26 serotype was highly resistant (75%) to 9 out of 12 different antibiotics tested.