Background: Helicobacter pylori is a Group 1 carcinogen because of its causal relationship to gastric carcinoma. We examined the role of drinking water as a potential mechanism of transmission in Peru, where gastric carcinoma is the most common cancer and cause of cancer death. Materials and Methods: Patients from high and low gastric cancer risk districts within Lima were enrolled to the study. Patients with histologically proven H. pylori infection were registered to the treatment and in-home water sampling portions of the trial. Gastric biopsies, filtered water, and biofilm specimens were analyzed for H. pylori by culture and quantitative polymerase chain reaction. Eradication was assessed by urea breath test at 6-8 weeks and one year from registration. Results: A total of 109 patients (89 from high-risk and 20 from low-risk districts) tested positive for H. pylori and were registered in the trial. Eradication rate by urea breath test at 6-8 weeks was 66%. Twenty-three patients who tested positive at 6-8 weeks responded favorably to second-line treatment (74.1%). Thus, the overall response rate at one year was 85%. H. pylori from 13 patients who failed to respond at 6-8 weeks demonstrated in vitro antibiotic resistance to amoxicillin, levofloxacin, and metronidazole. There was evidence of reinfection in five patients. Attempts to culture H. pylori from drinking water failed. H. pylori was detected by quantitative polymerase chain reaction in 97% of gastric mucosa, 50% of drinking water, and 30% of water biofilm specimens. Conclusions: We detected H. pylori by polymerase chain reaction in Lima’s drinking water, suggesting water as a source of infection (ClincialTrials.gov number NCT015128).