Background Urinary tract infection (UTI) is one of the most prevalent infectious diseases with worldwide health threatening. Antimicrobial resistant strains of Escherichia coli (E. coli) are a common cause of UTI which were identified as a treatment challenge. This study aimed to assay the prevalence of common β-lactam resistance genes including blaTEM, blaSHV, blaCTX-M and blaCMY and phenotypic resistance to commonly used β-lactam and fluoroquinolone antibiotics in UTIs. These factors were evaluated in various phylogenetic groups (phylotypes) of E. coli isolates. Real-time PCR was applied to detect β-lactam resistance genes and conventional PCR was used to determine the phylotypes. Phenotypic resistance against β-lactams (ceftazidime, cefotaxime, aztreonam and ceftriaxone) and fluoroquinolones (ciprofloxacin) were identified by the disc diffusion technique. The ability of extended spectrum β-lactamases (ESBLs) production in E. coli isolates was detected using the combined disc diffusion method. Results The prevalence of resistance genes were 89.6% for blaTEM, 44.3% for blaCTX-M, 6.6% for blaSHV and 0.9% for blaCMY. The two high prevalent phylotypes were B2 (29.2%) and D (17.9%) followed by E (14.1%), F (9.4%), C (6.6%) and 10.3% of isolates were unknown in phylotyping. Disc diffusion results showed high prevalence of antibiotic resistance to cefotaxime (88.6%), aztreonam (83%), ceftireaxon (77.3%), ceftazidime (76.4%) and ciprofloxacin (55.6%). Totally, 52.8% of isolates were found as phenotypical ESBL-producers. Conclusions This study’s results confirmed an explosion of antibiotic resistance amongst E. coli isolates from UTI against β-lactams and fluoroquinolones. Findings explain the necessity of deep changes in quantity and quality of drug resistance diagnosis and antibiotic therapy strategies. More studies are suggested to better and confident evaluations.