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Background: The eradication rates of sequential therapy are high in clinical trials; however, the adherence for follow‐up or the patient population in a real‐world setting might be different from those in trails. This study investigates the effectiveness of sequential therapy in a real‐world setting and the factors that lead to treatment failure. Materials and Methods: In this retrospective study, patients receiving sequential therapy as a first‐line anti‐Helicobacter pylori (H. pylori) treatment in a real‐world setting were reviewed. The age adjusted Charlson Comorbidity Index (age‐CCI) and baseline variety of medications were reviewed to determine factors correlated with nonadherence for post‐treatment testing and H. pylori eradication failure. Results: A total of 1053 patients were reviewed. A total of 579 patients receiving sequential therapy were included in the analyses. Among them, 462 received post‐treatment testing and were placed into the follow‐up group. Thus, the post‐treatment testing rate was 79.8%. Stroke was an independent factor of nonadherence for post‐treatment testing. In the follow‐up group, the eradication failure rate was 8.2%. Female sex (odds ratio [OR] 2.41 [95% CI 1.16–5.03], p = 0.02) and age‐CCI ≥2 (OR 3.16 [1.05–9.48], p = 0.04) were independent factors of H. pylori eradication failure. The eradication failure rates were 14.4%, 7.8%, 7.1%, and 3.1% for the females with age‐CCI ≥2, females with age‐CCI <2, males with age‐CCI ≥2, and males with age‐CCI <2 subgroups, respectively (p = 0.027). Conclusions: In a real‐world setting, the adherence rate of post‐treatment testing for sequential therapy as a first‐line anti‐H. pylori treatment was found to be suboptimal. Female sex and age‐CCI ≥2 were independent factors of eradication failure. [ABSTRACT FROM AUTHOR]