WFQ-101 with a unique N -1 substituent, 5-amino-4-fluoro-2-(hydroxymethyl)phenyl group, was selected as a lead compound through combination screening based on antimicrobial activity and the efflux index against quinolone-resistant (QR) Pseudomonas aeruginosa ( P. aeruginosa ). Through structural optimization, we identified WFQ-228 as a novel fluoroquinolone antibiotic candidate. WFQ-228 had potent and superior activity in comparison to levofloxacin (LVX) and ciprofloxacin (CIP) against clinical isolates of P. aeruginosa , Escherichia coli and Acinetobacter baumannii , including QR strains. Furthermore, WFQ-228 demonstrated the potential to overcome major mechanisms of drug resistance; its antimicrobial activity was less affected by both pump-mediated efflux and mutations of the quinolone resistance-determining region in P. aeruginosa compared with LVX and CIP. These results suggest that WFQ-228 is a promising candidate for further evaluation in the treatment of infections caused by QR Gram-negative pathogens.