Objectives: Limited data are available from recent trials involving pregnant women to guide Helicobacter pylori infection diagnosis. There are no data about the presence of H. pylori in the amniotic fluid as well. Furthermore, the relation between amniotic fluid H. pylori and hyperemesis gravidarum (HG) has not been characterized yet. Materials and Methods: This is a prospective study conducted after obtaining approval from the Ethics Committee. Pregnant women undergoing amniocentesis were enrolled in the study. The stool antigen test assessed the presence of H. pylori in amniotic fluid. A perinatologist independently performed an amniocentesis. The obtained amniotic liquid was sent to the laboratory to evaluate H. pylori infection by stool H. pylori antigen assay. We determined the rate of H. pylori in amniotic fluid and assessed relations between H. pylori infection and pregnancy outcome, including HG. Results: Between May and September 2017, we enrolled 48 pregnant women who underwent amniocentesis to detect possible fetal malformations. Patients were divided into two groups regarding the HG status. There were significant differences between the groups in terms of H. pylori infection presence. Among them, 28 (58.3%) were found to have a positive H. pylori test in their amniotic fluid. The rate of HG was significantly higher (71.4%) in patients who tested positive for H. pylori in amniocentesis than the H. pylori-negative group (20%), (p<0.001). Conclusions: The study's main new finding is that presence of H. pylori in the amniotic fluid is possible. Our data suggest that H. pylori-infected amniotic fluid is associated with the experience of past HG. The current study may have important implications for HG detection and help identify patients who would benefit from future preventive strategies.