The study investigated the frequency and antimicrobial susceptibility profiles of uropathogenic E. coli from pregnant women. Three hundred and thirty (330) mid-stream urine samples from pregnant women attending 2 antenatal clinics in the Onitsha metropolis were bacteriologically analyzed for the selective isolation of E. coli using standard techniques. Antibiotic susceptibility testing was carried out using the modified Kirby-Bauer disk diffusion technique. ESBL production was phenotypically detected using combined disk diffusion techniques, and confirmed using PCR technique. Of the 330 urine samples, 102 (30.9%) E. coli isolates were isolated. The prevalence of uropathogenic E. coli among the study volunteers was highest in the age group 26–35 years (37.9%), followed by those in 3rd trimesters (35.6%). A significant frequency of E. coli was recovered from business women (39.4%), multiparous (48.6%), and those with a history of UTI infection (48%). Antibiotic susceptibility tests showed that the most effective antibiotics against the strains were ofloxacin (58.8%) > gentamicin (57.8%) > nitrofurantoin (54%) > ciprofloxacin (51%) and cefixime (49%). Fifty-three (52%) isolates were multidrug resistant whereas 66.7% had a multiple antibiotics resistance index of > 0.2. Sixty-nine (67.6%) isolates were potential ESBL producers while 21(30.4%) isolates were confirmed ESBL producers. PCR results revealed that the uropathogenicE. coli harbored the blaTEM (66.7%) and blaSHV (38.1%) genes. A 30.9% prevalence of uropathogenicE. coli was detected and was significantly associated with the participant’s age, gestation, occupation, education level, UTI history, and parity at P-value < 0.05. blaTEM was the most predominant ESBL gene detected.