The minimum inhibitory concentrations (MICs) of 330 nonduplicate Helicobacter pylori isolates to nemonoxacin, tigecycline, and eight other antimicrobial agents were determined by using the agar dilution method. Sequencing the quinolone resistance-determining regions (QRDRs) in the gyrA gene of these isolates was also performed. Resistance to clarithromycin showed an increasing trend during the ten-year study period and was highest (38%) in 2005. Tigecycline had potent in vitro activities against all isolates, with an MIC(90) of 0.06 μg/ml. Among the quinolones tested, nemonoxacin (MIC(50) of 0.12 μg/ml and MIC(90) of 0.25 μg/ml) and gemifloxacin had one to two-fold better in vitro activities than ciprofloxacin, levofloxacin, and moxifloxacin. Among the nine isolates (2.7%) with levofloxacin resistance, four (44.4%) were also resistant to metronidazole, three (33.3%) to clarithromycin, and two (22.2%) to amoxicillin. Isolates with levofloxacin resistance exhibited one or two of three amino acid alterations (Ser-70, Asn-87, and Asp-91) involved in QRDRs in the gyrA gene. A double mutation at Ser70Cys and Asn87Ile had a higher level of resistance. The results of this study suggest a potentially useful role of nemonoxacin and tigecycline in the treatment of infections caused by H. pylori. The gyrA mutation at Ser-70 is a novel finding and has an impact on levofloxacin resistance.