Treatment of cystitis in primary care is usually empirical, guided by the prior probability of causal pathogens and their susceptibility. To re-evaluate empirical treatment guidelines, the actual distribution and susceptibility of uropathogens was examined and compared with two previous surveys in Belgium over the past 20 years. Because of the alarming increase in carriage of extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing Escherichia coli, this specific resistance was explored. From May 2014 to December 2015, 120 general practitioners collected midstream urine specimens from adult pre- and postmenopausal female patients with suspected cystitis. A dipslide was inoculated and sent for microbiological analysis. Anal swabs were collected for ESBL and carbapenemase detection. Of 265 enrolled patients, 203 (79.3 %) had a positive culture. Escherichia coli (81.6 %) was the most frequently isolated uropathogen, followed by Staphylococcus saprophyticus (8 %), confirming the results of the 1995 and 2005 surveys. The susceptibility of E. coli remained nearly 100 % for nitrofurantoin and fosfomycin, decreased from nearly 100 % in 1995 to 94.2 % for quinolones, from 73.2 to 55.5 % for ampicillin, and from 83.3 to 76.3 % for trimethoprim-sulfamethoxazole (TMP-SMX). In E. coli present in positive urine cultures, ESBLs were found in 2.5 % and carbapenemases were absent. In fecal specimens, ESBL-producing E. coli were found in 7.9 % and carbapenemases were not detected. Over a 20-year period, the distribution of uropathogens in women with cystitis remained unchanged. Susceptibility remained excellent for nitrofurantoin and fosfomycin. For TMP-SMX, ampicillin, and quinolones, there was a decrease.