Patients from a french hospital were infected by Salmonella enterica serotype Brandenburg multidrug-resistant strains including high-level resistance to fluoroquinolones. The characterisation of resistance mechanisms in a panel of 27 strains isolated between 1992 and 2013 is reported. Genetic relatedness of the isolates was determined by XbaI pulsed field gel electrophoresis and multilocus sequence typing. Fluoroquinolone target gene mutations were identified by sequencing the QRDRs of gyrA, gyrB, parC, parE genes PCR products. Mutations involved in increased efflux were identified by sequencing the following regulatory loci or genes of efflux pumps: ramRA, soxRS, marOR, acrR, and acrS. Role of the mutations identified were confirmed by complementing with the respective wild-type genes, MIC determinations and analysis by qRT-PCR of expression of efflux genes affected. All clinical strains studied were ST20 and grouped in a cluster, indicating a clonal population. The ciprofloxacin MICs showed increase over time and from 2000 ranged from 8 to 64 mG/L. Regarding target gene mutations several situations were observed: - one modification in GyrA and ParC (0.25-0.5 mG/L), and one in GyrB (2 mG/L); - two modifications in GyrA and one in ParC (8-32 mG/L), and one in ParE (32 mG/L); - two modifications in GyrA and ParC (64 mG/L). The diversity of the ciprofloxacin MICs levels in strains carrying the same target modifications and the ciprofloxacin MICs decrease (2 to 4-fold) in presence of the PABN efflux pump inhibitor, confirmed the role of the active efflux mechanism. High and variable expression levels of ramA and acrA were observed in all multidrug-resistant strains relative to a susceptible strain. Complementation confirmed the role of a RamR modification (Ala40Thr) which was responsible for increased expression of ramA (7 to 108-fold) and acrA (2 to 7-fold). In addition, a deletion in the RamR binding site resulted in increased expression of ramA (20-fold) and acrA (10-fold). An IS1 insertion sequence inactivating acrR, or the deletion of the acrR codon 300 resulted in increased expression of acrA (5 to 11-fold). In conclusion, various novel ramR and acrR mutations, responsible for increased efflux, were detected in the epidemic serotype Brandenburg ST20 clone.