Abstract Background Quinolone resistance (QR) is one component of the MDR emerging in Escherichia coli and is of particular concern given the widespread use of fluoroquinolones. Objectives To characterize the QR phenotypes and genotypes in E. coli responsible for bloodstream infections and to propose molecular determinants that could be targeted to predict ciprofloxacin resistance. Methods E. coli isolates from blood cultures in three French hospitals were studied for quinolone MICs and characterization of genotypic QR determinants (QRg). Results Among 507 isolates tested for MICs, 148 (29.2%) were resistant to quinolones based on EUCAST breakpoints and 143 (28.2%) harboured at least one QRg. QRg were mainly mutations in the QRDR (138 isolates, 27.2%), with 55.8% of these isolates carrying at least three QRDR mutations. gyrA mutations predominated (92.8%) followed by parC (61.6%), parE (32.6%) and gyrB (1.4%) mutations. Only 4.7% of the isolates harboured a plasmid-mediated quinolone resistance (PMQR) gene: aac(6′)-Ib-cr (60.0%) or qnr (qnrS, qnrB) (32.0%). For the first time in France, we reported the qepA4 allele of the plasmid-encoded efflux pump QepA. Only five isolates carried PMQR without a QRDR mutation. The positive predictive value (PPV) for ciprofloxacin resistance was 100% for any QRg and 99.2% for gyrA mutations specifically. Conclusions QR observed in E. coli isolates involved in bloodstream infections is still mainly due to QRDR mutations, especially at codons GyrA83/87, which could be used as a molecular target to rapidly detect resistance.