Helicobacter pylori colonizes more than 50% of the world population thus, it is considered an important cause of gastric cancer. The aim of this study was to determine the isolation frequency of H. pylori in Southern Chile from patients with symptomatology compatible with gastritis or gastric ulcer and to correlate these findings with demographic parameters of infected patients and the susceptibility profiles of the isolated strains to the antimicrobial drugs used in the eradication treatments. A total of 240 patients were enrolled in the study. Each gastric biopsy was homogenized and seeded onto blood agar plates containing a selective antibiotics mixture (DENT supplement). Plates were incubated at 37° C in a microaerophilic environment for five days. The susceptibility profiles to amoxicillin, ciprofloxacin, clarithromycin, tetracycline and metronidazole were determined using the E-test method. H. pylori was isolated from 99 patients (41.3%) with slightly higher frequency in female (42% positive cultures) than male (40.2% positive cultures). With regard to age and educational level, the highest isolation frequencies were obtained in patients between 21-30 (55%) and 41-50 (52.6%) years old, and patients with secondary (43.9%) and university (46.2%) educational levels. Nineteen (21.6%) strains showed resistance to at least one antimicrobial drug. Tetracycline was the most active antimicrobial in vitro, whereas metronidazole was the less active. One strain (5.3%) showed resistance to amoxicillin, clarithomycin and metronidazole, simultaneously.