The aim of this study was to assess the pattern of evolution of resistance to antibiotics in Helicobacter pylori isolated from children who underwent upper endoscopy with antral biopsy during a 10-year period (2001-2010). We retrospectively analyzed data of all children (n = 3,008) who underwent upper endoscopy during the observed period at the Children's Hospital Zagreb, a university tertiary medical center. We calculated the rate, antibiotic susceptibility and risk factors for the H. pylori infection in our cohort. Antral biopsy was performed in 2,313 (76.89%) patients. Altogether, 382 (16.51%) children had positive biopsy for H. pylori (histology and/or culture). There was no significant difference in the incidence of H. pylori during 10 years of observation (p = 0.21). Infected children compared to non-infected group were older (p = 0.005), and had more often antral nodularity (p < 0.0001), and duodenal ulcer (p = 0.002). Altogether, 22.4% of treatment-naïve patients had strains resistant to tested antibiotics: majority to azithromycin (17.9%), followed by clarithromycin (11.9%), metronidazole (10.1%) and amoxicillin (0.6%). In the eradication failure group, 9/11 of children had strains resistant to tested antibiotics, mostly to metronidazole (7/11), followed by azithromycin (3/11) and clarithromycin (1/11). No correlation was found between age or gender and antibiotic resistance (p = 0.32, for both). In conclusion, our data strongly support current guidelines which recommend antibiotic susceptibility testing prior to eradication therapy. Based on our results we recommend the use of amoxicillin-metronidazole-based regimen as the first-line therapy in our study population.