Delafloxacin is a new fluoroquinolone agent that has been introduced into clinical application. In this study, efficacy of delafloxcin on clinical isolates of Escherichia coli and Klebsiella pneumoniae was evaluated. Altogether, 80 E. coli and 43 K. pneumoniae were included in this study in Budapest, Hungary. Antimicrobial susceptibility testing was performed by broth microdilution for ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, and imipenem. Selected multidrug-resistant and delafloxacin resistant strains were further analyzed by whole-genome sequencing (WGS) on Illumina MiSeq platform to detect resistance determinants and sequence types (ST). Based on susceptibility results of E. coli, delafloxacin and ciprofloxacin resistance rates were 38.75% (31/80) and 41.25% (33/80), respectively. In the case of K. pneumoniae, delafloxacin and ciprofloxacin resistance rates were 46.5% (20/43) and 51% (22/43), respectively. WGS analysis detected mutations of quinolone resistance determining regions (QRDR) in selected delafloxacin resistant E. coli strains; namely, mutations in gyrA Ser83Leu, Asp87Asn and parC Ser80Ile were detected in ST162, ST57 and ST15840 strains. In the case of ST131 strains, five combined mutations were detected: gyrA Ser83Leu, Asp87Asn, parC Ser80Ile, Glu84Val, and parE Ile549Leu. In ST43 strains, gyrA S83L, D87N, parC S80I, and E84V mutations were detected. In delafloxacin-resistant K. pneumoniae, gyrA Ser83Ile and parC Ser80Ile mutations were detected in ST307 and ST147 strains. In the ST377 strain, gyrA Ser83Tyr, Asp87Ala, and parC Ser80Ile mutations were detected. Each delafloxacin-resistant K. pneumoniae strain carried OqxAB and AcrAB efflux pumps. Delafloxacin demonstrates efficacy against certain populations of E. coli and K. pneumoniae. However, multiple QRDR mutations can confer delafloxacin resistance in E. coli and K. pneumoniae clinical isolates.