There is evidence that infection by H. pylori can have a critical proportion in the development of hepatocyte injury and both noncancerous and malignant liver conditions including non-alcoholic fatty liver disease (NAFLD). This is attributed to several mechanisms, the most important one being the toxic products of the bacterium H. pylori and oxidative injury for hepatocytes which promotes hepatic injury. The present research was aimed at determining the association between H. pylori infection and the prevalence of NAFLD in Birjand, Iran. Two groups were included in this cross-sectional study at the outpatient university clinic. One group had NAFLD (65 patients) and the other group was healthy controls without NAFLD (65 subjects). The diagnosis of NAFLD was performed using abdominal ultrasound examination and the absence of taking steatogenic medications or alcohol. Serum anti-H. pylori IgG and fecal H. pylori antigen were tested for diagnosing of H. pylori infection using ELISA method. H. pylori infection diagnosis was made if both tests were positive. None of the subjects in either group had symptoms related to the digestive system including dyspepsia, GERD (gastroesophageal reflux disease), or epigastric pain suspicious of peptic ulcer disease. There were 37 patients (28.5%) in both NAFLD (22 cases, 33.8%) and control (15 cases, 23.1%) groups whose H. pylori tests (both IgG and fecal antigen) were positive. Statistically, no significant difference was observed between the two studied groups regarding H. pylori infection frequency (p = 0.37). Asymptomatic H. pylori infection rate was not significantly different between NAFLD patients and control subjects in Birjand, Iran.