Objective: It is reported that family members and sources of drinking water are potential routes of transmission for Helicobacter pylori. No data is available for Libyan families’ residents in a rural region. Thus, the aim is to determine the prevalence of H. pylori among healthy families. Materials and Methods: The study involved 119 healthy members of 30 families living in Sabratha city. A blood sample of 119 family members (61 males, 58 females, median age 35 years), using ELISA to detect anti-H. pylori IgG, a questionnaire was completed by interview. In addition, a total of 30 water samples were collected from the upper well level of the wells, and H. pylori-specific DNA was examined using PCR. Results: Overall seroprevalence of H. pylori was 47.45%, less in female than male participants (45%), 55%, respectively, a gradual increase with age, with the highest age group of 30-35 years. It found that eleven families (37%) have more than one member who is infected. H. pylori DNA was not found in well water samples with negative PCR results. Conclusions: The prevalence of H. pylori infection among the asymptomatic population in Sabratha city is high. PCR is an accurate and reliable method to identify H. pylori DNA. The well water in the natural environment in the rural region of Sabratha could not be a risk factor for H. pylori transmission or infected family members. member could play a role in the transmission of H. pylori in a Sabratha. However, further community-based research in other regions is necessary to identify risk factors associated with H. pylori infections.