BACKGROUND: Helicobacter pylori treatment failure remains a challenging problem. This study aimed to identify predictive factors for successful eradication in patients following treatment failures. METHODS: This was a retrospective cohort study. This study included 1,050 dyspeptic patients diagnosed with H. pylori infection at tertiary care center in Thailand between March 2014 and October 2021. Patients' demographic data, endoscopic findings, H. pylori culture, antimicrobial susceptibility testing (AST), treatment regimens and outcomes were analysed. RESULTS: Of 1,050 patients with H. pylori infections, 302 (28.7%) experienced treatment failure (mean age 58.4 years; 44.7% males). AST was performed in 192. Resistance was observed for metronidazole (43.2%), levofloxacin (33.9%), clarithromycin (24%), and amoxicillin (2.1%). There was no tetracycline resistance. Multidrug-resistance (MDR) was significantly more common following treatment failure (45.5% vs. 15.7%, p<0.001). Baseline characteristics were similar between treatment successes and failures. Eradication rates after first-line and second-line regimens were 71.2% and 54.5%, respectively. Medication nonadherence [OR 36.6 (95%CI 8.65-155.03, p<0.001)] and MDR [OR 4.49 (95%CI 2.29-8.81, p<0.001)] were associated with treatment failure. Over time, resistance increased for metronidazole, levofloxacin, and clarithromycin, while eradication rates with triple therapy declined. Tailored antibiotic therapy [OR 4.92 (95%CI 1.61-14.99, p = 0.005)] and a regimen including 4-times-daily dosing of amoxicillin (2 grams/day) [OR 3.05 (95%CI 1.10-8.41, p = 0.032)] were significantly associated with treatment success after first-line failure. Eradication rates when using tailored therapy and 4-times-daily dosing of amoxicillin (2 grams/day) were 91.1% and 89.4%, respectively. Performing AST before first-line therapy resulted in the highest cure rates. AST performed after multiple treatment failures was also associated with higher eradication rates compared with the group without AST (94.4% vs. 50%,p = 0.008). CONCLUSIONS: AST either before or after treatment failure correlated with a higher proportion of successful eradication. Nonadherence and the MDR infections predicted treatment failure. Tailored therapy and 4-times-daily dosing of amoxicillin after treatment failure were likely to be successful.