Plasmid-mediated quinolone resistance (qnr) genes confer low-level resistance but provide background for selection of highly-resistant strains. We investigated their prevalence and significance in clinical Enterobacteriaceae bacteremic isolates in Hong Kong. A prospective, hospital-based study was conducted (January 2008 to March 2009). Consecutive, non-duplicate blood isolates of extended-spectrum-β-lactamase (ESBL) and/or plasmid-mediated AmpC (PMAmpC) β-lactamase-producing Enterobacteriaceae were collected and subjected to qnr genes detection using multiplex PCR. Direct sequencing was performed to characterize the qnr and the co-existing bla genes. Clinical and microbiological variables, including antimicrobial resistance profiles, were compared between infections by ‘qnr-positive’ and ‘qnr-negative’ Enterobacteriaceae. Altogether 199 ESBL/PMAmpC-producing Enterobacteriaceae isolates were studied. qnr genes were detected in 20 % (qnrB, n = 24; qnrS, n = 16; qnrA, n = 0), of which 85% were Klebsiella spp. There was a strong association with PMAmpC genes (qnrB and DHA-1; p < 0.001). ‘qnr-positive’ isolates were more commonly hospital-acquired (60.0% vs 35.8%; adjusted OR 2.68, 95%CI 1.32–5.46) and multidrug-resistant (e.g. amoxicillin-clavulanate 90–100%, piperacillin-tazobactam 40–57%, ceftazidime 53–78%; sulfamethoxazole/trimethoprim 60–70%; ciprofloxacin 53–65%, levofloxacin 35–48%). Patients with ‘qnr-positive’ Enterobacteriaceae bacteremia had a higher 30-day mortality (45% vs 22%, p = 0.003). High Pitt bacteremia score, development of pneumonia, and failure to receive susceptible fluoroquinolone (adjusted HR 4.27; 95%CI 1.45–12.61) or carbapenem (adjusted HR 3.04; 95%CI 1.49–6.20) treatment were independent factors associated with death. A high proportion of ESBL/PMAmpC-producing Enterobacteriaceae (typically Klebsiella) bacteremic isolates carried qnr. These strains were multidrug-resistant, which was associated with inappropriate treatment and high fatality. Further dissemination of qnr and selection of fluoroquinolone/β-lactam-resistant strains should be closely monitored and controlled.