One hundred and six nalidixic acid-resistant Enterobacteriaceae isolates from two Brazilian hospitals isolated from June to October 2010 were evaluated to characterize the co-existence of plasmid-mediated quinolone resistant (PMQR) and extended-spectrum β-lactamase (ESBL) determinants. The qnr genetic environment was determined by PCR and sequencing. Conjugation and hybridization experiments determined whether qnr-carrying plasmids were self-transferable. The aac(6′)-Ib-cr and qepA genes were also screened. Thirteen qnr-like genes (12.3 %) were identified, with qnrB1 the most common, followed by qnrS1, qnrB2 and qnrB19. No qnrA, qnrC, qnrD or qepA determinant was detected. All qnr-positive strains possessed chromosomal substitutions in gyrase- and topoisomerase-encoding genes and four harboured a aac(6′)-Ib-cr gene. The co-production of blaCTX-M was observed in ten qnr-positive strains. These results indicate the dissemination of PMQR genes shown in clinical isolates from Brazil, and their co-existence with ESBL genes emphasizes the complexity of plasmid-mediated resistance determinants among Enterobacteriaceae.