Objectives: Helicobacter pylori is recognized as one of the most virulent microorganisms within the genus Helicobacter owing to its genomic characteristics. It has been associated with diseases, such as peptic ulcers and gastric cancer. This study aimed to evaluate Helicobacter pylori positivity in fecal samples using 16S rRNA gene polymerase chain reaction (PCR) in fecal samples. Methods: Fecal samples from patients with gastroduodenal symptoms were analyzed. Stool antigen test (SAT) was performed to identify H. pylori-positive cases. Subsequently, a pre-enrichment step was performed to increase the bacterial DNA yield and improve the molecular detection efficiency. Finally, PCR amplification, targeting a specific region of the H. pylori 16S rRNA gene, was performed to confirm the presence of the pathogen. Results: A 70% PCR positivity rate was observed among SAT-positive samples amplified for the 16S rRNA gene. Conclusions The positivity rate for H. pylori (70%) was confirmed by PCR, indicating that molecular testing of fecal samples is an effective non-invasive tool for detecting infection.