Objectives: This study aimed to assess the efficacy and safety of probiotics for Helicobacter pylori (H. pylori) eradication therapy as well as their effects on gastric microbiota.Methods: Patients who had failed H. pylori eradication therapy for at least twice were prospectively enrolled during March 2019 and March 2021. A 2-week administration of compound Lactobacillus acidophilus probiotic (1 g thrice daily) followed by a 10-day quadruple bismuth-containing H. pylori eradication therapy was administrated. Endoscopy was performed, and gastric biopsy samples were obtained for drug sensitivity testing and 16S rRNA gene sequencing before and after probiotic treatment. Eradication was evaluated by the 13 C-urea breath test at least 4 weeks after treatment completion. Adverse events (AEs) were recorded.Results: Thirty-seven patients were included. Probiotic supplementation had no effects on the diversity, community structure, and composition of gastric microbiota and no inhibition on H. pylori activity. However, it increased some of the beneficial bacteria such as Blautia, Dorea, and Roseburia (P < 0.05). The overall eradication rate was 97.3% (95% confidence interval [CI] 91.8%-100%). AEs, mainly dizziness, nausea, diarrhea, and chest distress, were detected in six individuals, all of which were resolved upon cessation of antibiotic administration. Dyspeptic symptoms were improved after probiotic supplementation and at treatment completion (both P < 0.001).Conclusions: H. pylori-infected individuals might benefit from probiotics followed by a quadruple bismuth-containing eradication therapy. Further studies with large sample sizes are warranted.