Introduction: Infection with Helicobacter pylori is one of the most frequent chronic bacterial illnesses in humans, infecting more than half of the world's population. Pregnant mothers are the populations most vulnerable to Helicobacter pylori infection. The frequency of H. pylori infection during pregnancy varies greatly depending on where you live. The objective of his study was to assess the prevalence of Helicobacter pylori infection and associated factors among pregnant mothers following antenatal care at antenatal care clinic in Debre Tabor General Hospital, Debre Tabor, Ethiopia, 2021. Methods and materials: Institutional based cross-sectional study design was conducted. Bivariate and multivariate logistic regression analyses were carried out to assess potential factors with Helicobacter pylori infection. Variables associated with the prevalence of Helicobacter pylori infection with (p-value < 0.05) were considered as significant factors. Results: The prevalence of Helicobacter Pylori infection was 52(17.9%), with 95% (CI: 13.4 – 22.3%). Sixty-five point four percent (65.4%) of Helicobacter Pylori infection positive mothers had gastrointestinal problems and developed symptoms like abdominal cramp and diarrhea 01 (1.92%), loss of appetite10 (19.23%), hiccups 9(17.31%), abdominal pain i.e. worsen when the stomach is empty 19(36.54%), indigestion and frequent heart burn13 (25%) during the present pregnancy. Lesser frequency of hand washing practice AOR= 3.09, 95%CI (1.14-8.34). Use of soap for hand washing AOR=0.44, 95% CI (0.19 - 0.98), feeding unwashed vegetables and fruits AOR=2.279, 95%CI (1.03-5.04), not being anemic AOR =0.268, 95% CI (0.10- 0.71), were significantly associated with Helico bacter pylori infection. Conclusions: This study shows that the prevalence of H. pylori infection was not low. The levels of anemia which was strongly associated with the prevalence of H. pylori infection. This implies that Helicobacter Pylori infection is a public health problem.