The prevalence of plasmid-mediated quinolone resistance genes [qnr, aac(6′)-Ib-cr and qepA] was sought among Enterobacteriaceae strains obtained from the Children’s Hospital of Tunis (Tunisia). Non-duplicate isolates (n = 278) with resistance to extended-spectrum cephalosporins and collected in 2003, 2007, 2008 and 2009 were screened for qnr genes. Forty (14.4 %) isolates were qnr positive and were screened for the presence of the aac(6′)-Ib-cr and qepA genes. qnrB was detected in 21 Klebsiella pneumoniae, 11 Escherichia coli and 6 Enterobacter cloacae isolates. Sequence analysis of the qnrB amplicons revealed variants including 24 qnrB1, 11 qnrB2 and 3 qnrB6. qnrS (qnrS1 allele) was detected only in K. pneumoniae isolates, either alone (two isolates) or with the qnrB gene (one isolate). The qnrA, qnrC and qnrD genes were not found in any of the 278 isolates. No qnr-positive isolates carried the qepA gene. Pyrosequencing results showed that aac(6′)-Ib-cr, a variant of the aac(6′)-Ib gene, was present in 31 qnr-positive isolates (21 K. pneumoniae isolates, seven Escherichia coli isolates and three Enterobacter cloacae isolates). aac(6′)-Ib was also found either alone (two isolates) or in association with aac(6′)-Ib-cr (one isolate). Of the 40 qnr-positive isolates, 92.5, 82.5, 57.5, 85 and 82.5 % were non-susceptible to nalidixic acid, ciprofloxacin, levofloxacin, ofloxacin and norfloxacin, respectively, and all were extended-spectrum β-lactamase producers. Random amplified polymorphic DNA-PCR typing of these isolates showed 16, 8 and 5 different genotypes in K. pneumoniae, Escherichia coli and Enterobacter cloacae isolates, respectively. Our study highlights the high prevalence of qnr in association with aac(6′)-Ib-cr among Enterobacteriaceae isolates, even from children, who are patients not overtreated with quinolones.