Avian pathogenic Escherichia coli causes high mortalities and substantial economic losses in the poultry industry worldwide, a problem that intensified with the increasing antibiotic resistance. In this study, we characterized fluoroquinolones resistance in E. coli from broiler chickens with colibacillosis. A total of 150 visceral organs collected from broiler chickens with postmortem colibacillosis lesions were subjected to bacteriological and biochemical examination. Out of them, 89 samples (59.3%) were found positive for E. coli. The most predominant serogroups were O125 (23.6%), O44 (16.6%), O127 (15.7%), and O18 (11.2%). The antimicrobial susceptibility testing of the recovered E. coli isolates against seven quinolone antibiotics revealed that 88.8% of the isolates were resistant to at least one tested antibiotic, while 25.8% were resistant to all of them. The highest resistance rates were observed against flumequine (77.5%) and nalidixic acid (73%), while the lowest resistance rate was observed against levofloxacin (29.2%). A total of 12 isolates displaying phenotypic resistance to fluoroquinolones were then screened for plasmid-mediated quinolone resistance (PMQR) genes qnrA, qnrB, qnrS and aac(6′)-Ib-cr by PCR. The results showed that 66.7% of the isolates were positive for qnrS gene, while aac(6′)-Ib-cr, qnrA and qnrB genes were not detected in any isolate. The high occurrence of fluoroquinolones resistance and fluoroquinolones resistance genes in avian pathogenic E. coli is alarming, and urgently requires effective monitoring of the use of fluoroquinolones in the broiler sector, with regular screening for these resistance genes to evaluate the scale of their threat to veterinary practice and public health.