Objective The aim of this study was to evaluate the efficacy/tolerability of a culture-guided approach in the eradication of Helicobacter pylori and identify factors associated with antibiotic resistance/treatment failure. Patients and methods This retrospective single-center study included patients who underwent culture-guided treatment for H. pylori infection, after two ineffective eradication attempts, between October 2012 and December 2016. We assessed the following demographic and clinical data of the patients: sex, age, BMI, alcohol and tobacco consumption, history of dyspepsia, peptic ulceration and first-degree relatives with gastric cancer, antibiotic susceptibility results, treatment composition, tolerability, and success. The treatment success was confirmed by a monoclonal stool antigen test. Results Culture-guided treatment was performed in 42 patients (57% women, mean age +/- SD: 48.9 +/- 11.4 years). The rates of antibiotic resistance were as follows: clarithromycin 86%, metronidazole 67%; levofloxacin 52%, tetracycline 2%, and amoxicillin and rifampicin 0%. Double resistance to clarithromycin and metronidazole was found in 59.5% of the patients. Most patients showed resistance to less than three antibiotics, but 31% were resistant to three or more. Intention-to-treat and per-protocol eradication rates were 59.5 and 61.5%. Adverse events occurred in 15 (35.7%) patients, but only two (4.8%) patients did not complete treatment because of adverse events. Only age more than 50 years was associated with resistance to three or more antibiotics. Having a first-degree relative with gastric cancer was associated with treatment failure and having a BMI of at least 25 kg/m(2) protected from failure. Conclusion Third-line culture-guided treatment often fails to eradicate H. pylori infection. We need to find factors other than in-vitro antibiotic resistance to explain these suboptimal results. Copyright (C) 2017 Wolters Kluwer Health, Inc. All rights reserved.