Background: Unsuccessful treatment of H. pylori (HP) infection is constantly rising. Antibiotic susceptibility testing and selection of individualized therapy are expected to improve eradication of HP. Aim: To assess the diagnostic accuracy of microbiology testing for HP and to assess the effectiveness of culture-guided susceptibility tested antibiotic therapy. Materials and methods: A total of 62 HP infected patients, 35 with unsuccessful eradication and 27 treatment naive were subjected to culture testing and 3 endoscopy-based methods. In isolated НР colonies strain susceptibility and resistance were determined by disc-diffusion method. A triple therapy in the naïve group and quadruple in the unsuccessfully treated group was applied. The effectiveness of eradication was evaluated after 2 months by stool antigen test and/or urea breath test. Fisher’s exact test for comparison of therapeutic efficacy was used. Results: НР colonies were isolated in 13 patients - 9 naïve and 4 unsuccessfully treated. Higher sensibility (100%) was shown towards UBT and high specificity between 95% - 100% compared to other methods. Its accuracy was 91.9%. In all patients with proven resistance culture-guided eradication was successful. Conclusions: Microbiology and antibiotic susceptibility testing allows optimal diagnosis and therapy of HP infection.