Hospital-adapted Enterococcus faecium differ from their colonising variants in humans and animals by additional genomic content. Molecular typing based on multilocus sequence typing (MLST) allows allocation of isolates to specific clonal complexes (CCs), such as CC17 for hospital-adapted strains. Acquired ampicillin resistance is a specific feature of these hospital isolates, especially in Europe. A few recent reports have described acquired high-level ciprofloxacin resistance as a supposed feature of hospital-adapted E. faecium strains. In the present retrospective analysis, ciprofloxacin minimum inhibitory concentrations (MICs) of 609 clinical isolates from German hospital patients (1997-2007) were determined and a breakpoint for high-level resistance was deduced (>16mg/L). Acquired high-level ciprofloxacin resistance was distributed among isolates of 26 different MLST types (all CC17), indicating a wide prevalence of this acquired resistance trait among the hospital-adapted E. faecium population. High-level ciprofloxacin resistance was linked to gyrA and parC mutations in 98 investigated isolates. Eleven different allele types or combinations thereof were identified. Their allocation to specific MLST and pulsed-field gel electrophoresis (PFGE) types revealed differences in the emergence and spread of corresponding mutations and strains.