Resistance to ciprofloxacin in Escherichia coli from urinary tract infections (UTI) in Denmark is increasing parallel to increased use of fluoroquinolones both in Denmark and in other European countries. The objective was to investigate the occurrence of ciprofloxacin resistance mechanisms, phenotypic coresistance, and if ciprofloxacin resistance was caused by clonal spread or to individual mutational events in a collection of consecutively obtained E. coli submitted to a clinical microbiology department at a Danish hospital. One hundred four UTI-related E. coli resistant toward nalidixic acid by disc diffusion were typed by Pulsed Field Gel Electrophoresis (PFGE) with XbaI. One isolate representing each PFGE type and only one patient (n = 77) were investigated for point mutations in sequenced PCR amplicons of the four topoisomerase genes; qnr genes by use of PCR; aac(6')-Ib-cr by BtsCI restriction of PCR products; and efflux using efflux pump inhibitors in a broth dilution assay. Minimal inhibitory concentration (MIC) was determined for 21 antibacterial agents, including ciprofloxacin. Of the 77 isolates, the majority were resistant to ciprofloxacin (91%) and multiresistant (resistant to ≥ 3 antimicrobial classes, 83%). Ciprofloxacin-resistant isolates showed at least one target mutation. A significant, positive correlation was found regarding MIC of ciprofloxacin and the number of target mutations. Efflux was found as a resistance mechanism in 77% of isolates tested (n = 60). The aac(6')-Ib-cr gene was detected on plasmids from five isolates showing ciprofloxacin MICs >512 mg/L. No overall clonal relationship among isolates was found according to PFGE. Target modification is the dominating fluoroquinolone resistance mechanism often found in combination with efflux and sometimes aac(6')-Ib-cr. In Denmark, increasing ciprofloxacin resistance in E. coli is mainly due to mutational events and not to spread of clones.