Objectives To characterize the mechanisms of fluoroquinolone and cephalosporin resistance in Enterobacteriaceae from a Dutch teaching hospital in 2008. Methods We sequenced gyrA , gyrB , parC and parE. The presence of plasmid-encoded genes qnrA , qnrB , qnrS , aac(6′)-Ib , qepA , bla TEM , bla SHV, bla OXA , bla CTX-M and bla AmpC was studied by PCR. Escherichia coli isolates were further characterized by AFLP and multilocus sequence typing (MLST). Results In total, 49 E. coli , 16 Klebsiella pneumoniae and 3 Enterobacter cloacae isolates were investigated. Mutations in gyrA were found in all E. coli isolates. Forty-five (92%) E. coli isolates carried at least one point mutation in parC . Most E. coli isolates (59%) also carried mutations in parE , of which I529L was the most prevalent. I529L was unequivocally associated with E. coli sequence type (ST) 131. This single-nucleotide polymorphism (SNP) was later also found in eight out of nine ST131 strains from another collection. Twenty-nine E. coli isolates carried extended-spectrum β-lactamase (ESBL) genes, predominantly bla CTX-M-15 . In E. coli , aac(6′)-Ib-cr was the predominant plasmid-mediated resistance mechanism, whereas in K. pneumoniae qnr genes were found mostly. In K. pneumoniae isolates, qnr and aac(6′)-Ib-cr co-occurred with ESBL genes ( n = 13; bla CTX-M and bla SHV ) and/or bla AmpC ( n = 3; bla DHA-1 ). Conclusions E. coli ST131 was the predominant clone, which accumulated a high number of chromosomal mutations. The I529L SNP in parE was a signature of most, but not all, ST131 strains. In contrast to E. coli , fluoroquinolone resistance mechanisms were predominantly plasmid-encoded in K. pneumoniae .