This study investigated the association between prescription of fluoroquinolones and emergence of ciprofloxacin resistance among Escherichia coli isolates in the urine of outpatients from whom a ciprofloxacin-sensitive E. coli strain had been isolated previously. Patients were identified and followed using the healthcare databases of Emilia-Romagna Region, Italy. The outcome of interest was the first isolation from urine of an E. coli strain resistant to ciprofloxacin. Prescription of fluoroquinolones during the previous 6 months was associated independently with the emergence of ciprofloxacin resistance; the strength of the association varied according to individual fluoroquinolone agents.