E. coli is a major pathogen of commercially produced poultry all over the world, causing colibacillosis and contributing high significantly to economic losses. In this study, isolation, serotyping, virulence factors and antimicrobial susceptibility test were characterized for avian pathogenic E. coli strains that isolated from tissues of the infected pigeons and from non-hatched pigeon eggs in Assiut Province. 124 samples (87 pigeons and 37 non-hatched pigeon eggs) were examined for E. coli infection. The samples include liver and kidney tissues of diseased and freshly dead pigeons plus yolk of 20 infertile eggs and yolk sac of dead-in-shell embryos of the non-hatched pigeon eggs. Twenty three bacterial isolates were identified, from which 19 isolates for E. coli and one isolate for each Enterobacter agglomeranns, Enterobacter cloacae, Hafnia alvei and Serratia marcescens from tissues of infected pigeons while samples of pigeon eggs were found negative for E. coli isolation. Eight serogroups were identified among sixteen of pigeon E. coli isolates, however, 3 isolates were nontyped. The most common serogroup was O78 that identified in (21.05%) followed by serogroup O2:H6 (15.79%) among the E. coli isolates. Each serogroup of O1:H7, O128:H2 and O119: H4 were isolated by 10.53%, while each serogroup of O111:H4, O114:H21 and O44:H18 were represented with an incidence 5.26%. The characteristic virulence factors of hemolysis and Congo red binding activities of E. coli isolates were done. All E. coli isolates (100%) were found positive for the Congo red binding activity and (84.2%) of the isolates were positive for hemolytic activity. The in vitro antimicrobial sensitivity of the E. coli isolates were tested against 8 antimicrobial drugs used in pigeon treatment. Overall, the all E. coli isolates displayed resistance to neomycin at all (100%), the most strains were frequently highly resistance to trimethoprim-sulphmethoxazol followed by ampicillin, tetracycline, erythromycin and doxycycline. The isolates show high sensitivity to streptomycin (100%) and norfloxacine (89.5%). These findings suggest that multiple-antimicrobial-resistant of E. coli isolates are commonly present among infected pigeons that can be potentially transmitted from pigeons to humans. So the need for the introduction of surveillance programs to monitor antimicrobial resistance in pathogenic bacteria is recommended.