Helicobacter pylori resistance to standard therapeutic antimicrobials (clarithromycin, metronidazole, amoxicillin, and tetracycline) has been demonstrated, so other treatment options are urgently needed. Fluoroquinolones like levofloxacin (LVX) and gatifloxacin (GAT) have been evaluated as alternatives, but resistance associated with gyrA gene mutations affects their efficacy. Novel quinolones garenoxacin (GRNX) and sitafloxacin (STFX), more potent against gram-positive bacteria than LVX and GAT, are available, with STFX being the most active against H. pylori (MIC90, -0.008 mg/liter). This study compared the in vitro activities of GAT, GRNX, and STFX against 23 H. pylori strains with gyrA mutations isolated from patients with primary or secondary eradication failure. Susceptibility was determined by the agar dilution method per National Committee for Clinical Laboratory Standards guidelines, using previously reported gyrA mutation status confirmed by PCR sequencing. Results showed STFX and GRNX exhibited potent activity against gyrA-mutant strains: STFX had a MIC90 16-fold lower than GAT, GRNX 4-fold lower; STFX had a MIC50 16.6-fold lower than GAT, GRNX 4-fold lower. These findings suggest STFX and GRNX may overcome the resistance of H. pylori strains with gyrA mutations to previous quinolones, and switching to these quinolones may improve third-line H. pylori eradication efficacy.