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This study determined the antimicrobial resistance profiles and molecularly characterized beta-lactam- and fluoroquinolone-resistant E. coli recovered from human clinical samples. Three hundred (300) clinical samples collected from two major tertiary healthcare institutions were analyzed using standard microbiological techniques. Antimicrobial susceptibility testing and phenotypic detection of extended-spectrum beta-lactamase (ESBL) were done by disc diffusion and Double Disk Synergy Test. Molecular characterization for ESBL and fluoroquinolone-resistant genes were done by PCR with specific primers. In total, 44 (14.7%) ESBL-producing E. coli were recovered from human clinical specimens. The recovered isolates were multidrug-resistant with multiple antibiotics resistance index values ranging from 0.5–0.8. Isolates exhibited resistance to sulfamethoxazole/trimethoprim 44 (100%), cefoxitin 42 (95.5%), tetracycline 42 (95.5 %), cefuroxime 41 (93.2%), ciprofloxacin 40 (90.9%), cefotaxime 37 (84.1%), amoxicillin 37 (84.1%), amoxicillin/clavulanic acid 37 (84.1), and ceftazidime 35 (79.5%). BlaTEM 41 (93.2%) ESBL gene was the most predominant gene among the isolates, followed by blaCTX-M 9 (20.5%), and blaSHV 1 (2.3%) genes. Co-existence of blaTEM +blaSHV, blaTEM + blaCTX-M, and blaTEM +blaSHV + blaCTX-M genes was observed in 1 (2.3%), 7 (15.9%), and 4 (9.1%) isolates, respectively. All the fluoroquinolone-resistant isolates harbored aac-lb-6-cr gene while Qnr gene was absent. Association of fluoroquinolone resistance- and β-lactam resistance genes; aac-lb-6-cr + blaTEM + blaSHV + blaCTM was observed in 5 (11.4%) isolates. Our study showed high frequency of ESBL-producing E. coli harboring ESBL and fluoroquinolone resistance genes from human clinical samples in Ebonyi State, Nigeria. The high frequency of multidrug-resistant E. coli in our study is very worrisome and could have significant public health impact such as treatment failures, and possibly death, if not properly managed. It is therefore imperative to establish strong regulative measures and guidelines that would help in curtailing the increasing dissemination of these superbugs in healthcare institutions in Nigeria.