Avian colibacillosis is one of the most important diseases of chickens, resulting in high economic losses as well as high morbidity and mortality. In the current study, the prevalence of avian colibacillosis was studied in 200 broiler chickens obtained from different farms in Beni-Suef and El-Fayoum Governorates. A total of 200 pooling samples were collected aseptically from heart blood as well as the affected internal organs including airsacs, pericardial sac, liver, lung and ascetic fluids of slaughtered diseased and freshly dead broiler chickens. Bacteriological examination of the collected samples showed that a total of 58 E. coli isolates were recovered with a prevalence of 29%. The results of in-vitro antimicrobial susceptibility tests revealed that E. coli isolates showed high sensitivity to colistin sulphate only (72.4%). On the other hand, high resistances were recorded to all other antimicrobials including cefotaxime sodium and florophenicol (96.6% for each), apramycin, ciprofloxacin and gentamicin (91.4% for each), enrofloxacin and lincomycin (91.4% for each), streptomycin (89.7%), sulphamethoxazol-trimethoprim and doxycycline HCl (77.6% for each) and spiramycin (75.9%). Moreover, all E. coli isolates were MDR (100%). PCR was applied on 10 E. coli MDR isolates for detection of 7 genes; 4 resistance-associated genes (bla_TEM, tetA, sul1 and dfrA) as well as 3 virulence-associated genes (tsh, iss, iutA). The results revealed that 90% of isolates had at least 4 virulence genes while only10% had not any gene. The gene bla_TEM was the most prevalent (90%) followed by iutA and sul1 (70% for each) then, iss and tetA (60% for each). Meanwhile dfrA and tsh genes were represent in 40% and 30% of isolates, respectively.