Delafloxacin is a novel fluoroquinolone agent that is approved for clinical application. In this study, we analyzed the antibacterial efficacy of delafloxacin in a collection of 47Escherichia colistrains. Antimicrobial susceptibility testing was performed by the broth microdilution method and minimum inhibitory concentration (MIC) values were determined for delafloxacin, ciprofloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, and imipenem. Two multidrug-resistantE. colistrains, which exhibited delafloxacin and ciprofloxacin resistance as well as extended-spectrum beta-lactamase (ESBL) phenotype, were selected for whole-genome sequencing (WGS). In our study, delafloxacin and ciprofloxacin resistance rates were 47% (22/47) and 51% (24/47), respectively. In the strain collection, 46E. coliwere associated with ESBL production. The MIC50 value for delafloxacin was 0.125 mg/L, while all other fluoroquinolones had an MIC50 value of 0.25 mg/L in our collection. Delafloxacin susceptibility was detected in 20 ESBL positive and ciprofloxacin resistantE. colistrains; by contrast,E. colistrains that exhibited a ciprofloxacin MIC value above 1 mg/L were delafloxacin-resistant. WGS analysis on the two selectedE. colistrains (920/1 and 951/2) demonstrated that delafloxacin resistance is mediated by multiple chromosomal mutations, namely, five mutations inE. coli920/1 (gyrAS83L, D87N,parCS80I, E84V, andparEI529L) and four mutations inE. coli951/2 (gyrAS83L, D87N,parCS80I, and E84V). Both strains carried an ESBL gene,blaCTX-M-1inE. coli920/1 andblaCTX-M-15inE. coli951/2. Based on multilocus sequence typing, both strains belong to theE. colisequence type 43 (ST43). In this paper, we report a remarkable high rate (47%) of delafloxacin resistance among multidrug-resistantE. colias well as theE. coliST43 international high-risk clone in Hungary.