Background: Helicobacter pylori is a bacterium infection that is a risk factor for Peptic Ulcer Disease, and affected individuals remain asymptomatic until they present with complications. This study aimed at determining the prevalence and risk factors of Helicobacter pylori infection among patients with Peptic Ulcer Disease undergoing upper gastrointestinal endoscopy at Benjamin Mkapa Hospital. Methods: The study was a cross-sectional analytical Hospital-based study where a quantitative approach was used. A total of 149 patients with a Peptic Ulcer Disease with the age of ≥ of 18years were recruited at Benjamin Mkapa Tertiary and Teaching Hospital between February and April 2020. Demographic and clinical characteristics were captured by using a standard questionnaire. The patients’ association characteristics were tested by using the X2 with the corresponding p-value and risk factors measured by logistic regression, a p-value of<0.05 considered significant for the study at a 95%CI. Helicobacter pylori infection was detected using a monoclonal antigen test. Results: The overall prevalence of Helicobacter pylori infection in the study was 49.66 %. H. pylori infection was associated with age ≥60years (AOR=5.46, 95%CI (1.77-16.86) p-value=0.0032), Use of unsafe drinking water (AOR=2.51, 95%CI (1.11-6.22) p-value 0.0255). NSAID user (AOR=3.16, 95%CI (1.10-9.11) p-value 0.0330). Relatives with PUD (AOR=2.46, 95%CI (1.08-5.62) p-value 0.0323). Conclusion: The prevalence of Helicobacter pylori infection was found to be relatively high in this study. Helicobacter pylori infection was significantly associated with an increase in age, low level of education, unsafe water, Relatives with PUD, and the use of NSAIDS.