An alternative screening test is needed to efficiently eradicate Helicobacter pylori from a population with prevalent upper gastrointestinal lesions. We evaluated the performance of a new one-step fecal test for H. pylori for diagnosis of H. pylori infection in Taiwan. Methods: We developed a fecal test to detect H. pylori based on the immunochronomatographic assay and a mixture of monoclonal antibodies. We first recruited symptomatic patients from the primary care setting to evaluate fecal test performance using a reference standard consisting of 13C urea breath test, rapid urease test, and histology. We also compared the performance of the fecal test with that of others. Next, we recruited asymptomatic participants from the mass screening setting to evaluate population attendance for the fecal test and compared its performance with that of 13C urea breath test. Results: In the primary care setting, 117 patients were recruited; H. pylori infection was confirmed in 58 (49.6%). Fecal test sensitivity, specificity, positive and negative predictive values, and accuracy were 88.0% [95% confidence interval (CI): 79.6–96.4%], 100%, 100%, 89.4% (95% CI, 82.0–96.8%), and 94% (95% CI, 89.7–98.3%), respectively. Fecal test specificity and positive predictive value were significantly higher than those of the serological test, whereas the sensitivity and negative predictive value were lower than those of the 13C urea breath test (p < 0.05). In the mass screening setting, 2720 of 3520 invited individuals participated (77.3%; 95% CI, 76–78.7%); 649 (23.9%) showed positive results. Concordance rate and kappa statistic between the fecal test and 13C urea breath test were 91.7% (563/614; 95% CI, 89.9–94.1%) and 0.78 (95% CI, 0.73–0.84), respectively. Conclusion: Given the acceptable sensitivity, excellent specificity, and high participation rate to screening, the one-step H. pylori stool antigen test is feasible for wide application in the community.