In eradication of Helicobacter pylori infection, standard quadruple second-line therapy consisting of proton pump inhibitor (PPI), bismuth, metronidazole and tetracycline often fails and shows poor patient compliance. The aim of our study was to evaluate the efficacy and tolerability of moxifloxacin-based triple therapy as an alternative second-line protocol. A total of 160 patients, in whom the initial standard PPI triple therapy had failed to eradicate H. pylori infection, were included in the study. The initial H. pylori status was assessed using the (13)C-urea breath test. Patients were randomized to one of the following 7-day treatment regimens: (1) OMM: omeprazole 20 mg twice a day, moxifloxacin 400 mg/day, metronidazole 500 mg three times a day; and (2) OBMT: omeprazole 20 mg twice a day, colloidal bismuth subcitrate 120 mg four times a day, metronidazole 500 mg three times a day, tetracycline 500 mg four times a day. Patient compliance and adverse events were evaluated one week after completion of therapy. H. pylori status was re-assessed with the (13)C-urea breath test six weeks after the end of therapy. The eradication rates were 73.2% (60/82) and 78.9% (60/76) with moxifloxacin-based triple therapy, and 53.8% (42/78) and 64.6% (42/65) with bismuth-based quadruple therapy, by intention-to-treat (p = 0.018) and per-protocol (p = 0.088) analyses, respectively. Adverse events/intolerability were described in 12/82 patients in the OMM group and 18/78 patients in the OBMT group. Compliance with treatment was 92.7% in the OMM group and 83.3% in the OBMT group. Moxifloxacin-based triple therapy is a highly effective second-line eradication treatment in H. pylori infection. Because of its high levels of safety and tolerability, this protocol represents an adequate alternative to the standard bismuth-based quadruple therapy.