DNA sequencing and real-time PCR were used to evaluate the gyrA and parC mutations, AcrAB efflux pump over-expression, and their correlation with high-level resistance to fluoroquinolones in 74 fluoroquinolone-resistant clinical Escherichia coli isolates recently collected in Taiwan. RAPD analysis revealed high clonal diversity. Isolates with four to five mutations (especially Ser83Leu, Asp87Asn [or Asp87Tyr], and Ala93Thr in gyrA and Ser80Ile and Glu84Gly in parC) had increased resistance levels. The acrA gene was over-expressed in 51% of 74 resistant isolates. The trend was towards increased fluoroquinolone MICs in isolates with both multiple mutations in the quinolone-resistance determining region (QRDR) and over-expression of the AcrAB efflux pump. Furthermore, acrA gene over-expression was significantly correlated with cross-resistance to beta-lactams including piperacillin, amoxicillin, clavulanic acid, and cefazolin. In conclusion, mutations in the QRDR are the primary mechanism for increasing fluoroquinolone resistance, and in combination with efflux pump over-expression, contribute to high-level resistance.