INTRODUCTION: Multidrug resistance among Escherichia coli causing Urinary Tract Infections (UTIs) is a major public health problem, threatening the effective treatment of UTIs. This study investigated the phenotypic and molecular characteristics of E. coli associated with UTIs in Port-Harcourt, Nigeria. METHODS: Twenty-five non-duplicate isolates of E. coli from UTIs patients at the University of Port-Harcourt Teaching Hospital, Nigeria were identified using Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry. The antimicrobial susceptibility patterns were determined using Kirby-Bauer disc diffusion technique. Phenotypic expression of Extended Spectrum Beta Lactamases (ESBLs) and AmpC beta-lactamase were determined using standard laboratory methods and polymerase chain reaction (PCR) was used to detect ESBLs, AmpC, Quinolones and Aminoglycosides resistance genes. RESULTS: The isolates exhibited high rates of resistance to co-trimoxazole (76%), nalidixic acid (68%), ciprofloxacin (60%), gentamicin (44%) and low resistance to cefotaxime (20%) but were fully susceptible to cefoperazone/sulbactam, amikacin, nitrofurantoin, colistin and carbapenems. Phenotypic expression of ESBLs was recorded in 6(24%) isolates while genotypic detection revealed the highest prevalence of blaTEM 22(88%), followed by blaCTX-M-15 16(64%), blaSHV 7(28%) and blaOXA-1 6(24%) while AmpC (blaCMY-2) gene was detected in 8(32%) isolates. Amongst the quinolone resistant isolates, qnr variants (qnrB, qnrD and qnrS) and aac(6')-Ib genes were detected in 7(28%) and 3(12%) isolates respectively while all gentamicin resistant isolates possessed the aacC2 gene. The co-expression of blaCTX-M-15 with quinolones and aminoglycoside genes were 20% and 40% respectively. The prevalence of multiple drug resistance was 52%. CONCLUSION: A high proportion of the studied E. coli isolates co-expressed ESBLs, quinolones and aminoglycosides resistance genes which call for prompt antibiotic stewardship and preventive strategies to limit the spread of these genes.