Objectives To optimize empirical treatment of urinary tract infections (UTIs), regular evaluation of the antibiotic susceptibility of the most common uropathogen, Escherichia coli , is necessary. We compared the antibiotic prescription rate for UTIs in women and the E. coli antibiotic susceptibility results, including the prevalence of extended-spectrum β-lactamase (ESBL)-producing strains, in 2009 with data collected 5 years earlier. Patients and methods Urinary samples from female patients with symptoms of uncomplicated UTI in 42 general practices were collected over a 6 month period. Uropathogens were identified and the antibiotic susceptibility of E. coli was determined. Results We analysed 970 urine cultures, of which 785 (81%) were considered positive (≥103 cfu/mL). E. coli accounted for 72% of the isolates. ESBLs showed an increase between the two surveys (0.1% versus 1%; P < 0.001), while no difference in antibiotic susceptibility to the commonly used antimicrobial agents for UTIs was observed. A significantly lower susceptibility rate to co-amoxiclav was observed in the eastern region of the Netherlands, as compared with the northern region (80% versus 92%; P < 0.05). Consistent with national guidelines, the prescription rate of trimethoprim decreased over time (19% versus 5%; P < 0.05) whereas nitrofurantoin and fosfomycin rates showed an increase (58% versus 66% and 0% versus 6% respectively, both P < 0.05). Conclusions Over a 5 year period, the antibiotic susceptibility of uropathogenic E. coli did not change in female patients with uncomplicated UTI in the Netherlands, but ESBL prevalence increased. With respect to the prescription of antimicrobial agents, compliance with national UTI guidelines was good.