Resistance to antibiotics poses a major challenge, especially in cases of urinary tract infections attributed to Escherichia coli. The rise of extended-spectrum β-lactamase (ESBL)-producing and aminoglycoside-resistant strains complicates treatment. This research focused on identifying the occurrence of both phenotypically confirmed ESBL and AME genes in E. coli isolated from patients with UTIs in Sana’a, Yemen. This cross-sectional study encompassed a sample of 378 patients at Al-Kuwait University Hospital, Sana’a. Midstream urine samples were cultured and E. coli isolates identified via standard methods. Susceptibility to antimicrobial agents was determined through the Kirby-Bauer method, following the guidelines issued by CLSI in 2019. ESBL production was phenotypically detected and AME genes (aac(6′)-Ib, aac(3′)-IIa, aph(3′)-Ia, and ant(2”)-Ia) were detected via multiplex PCR. Among 378 samples, 167 E. coli isolates were identified (44.18%), of which 76 (45.5%) were ESBL-producers. AME genes were detected in 52.6% of ESBL isolates, with aac(6′)-Ib being most frequent (39.47%), followed by ant(2”)-Ia (31.58%) and aph(3′)-Ia (11.84%). Co-occurrence of ≥2 AME genes was seen in 36.4% of isolates. Risk factors for resistance included catheterization, hospitalization, and older age. The study identified a high rate of dual antimicrobial resistance among E. coli isolates in Yemen underscores the need for enhanced molecular surveillance and antimicrobial stewardship programs. Empiric therapies should prioritize amikacin and carbapenems in high-risk cases.