Helicobacter pylori (H. pylori, Hp) is a primary contributor to various stomach diseases, including gastritis and gastric cancer. This bacterium can colonize gastric epithelial cells, compromising their integrity and leading to the development of these conditions. While antibiotics are the mainstay of treatment for H. pylori infections, their widespread use has led to serious issues with drug resistance. High-temperature requirement A (HtrA) protein is an active serine protease secreted by H. pylori, which can destroy gastric epithelium, thus helping H. pylori to colonize gastric mucosa efficiently. In this study, we identified three compounds-Quercetin, Fisetin, and Geniposide-as potential natural compounds that might specifically interact with the HtrA protein, based on molecular docking and molecular dynamics simulations (MDs). The casein hydrolysis experiment indicated that Fisetin could inhibit the activity of HtrA in hydrolyzing casein at the concentration of 50 μM m. Additionally, our in vitro antibacterial experiments further showed that Fisetin could effectively inhibit the growth of H. pylori in a concentration-dependent manner, with an inhibition rate of 80% achieved at a concentration of 10 μM. In summary, these results suggest that Fisetin has an inhibitory effect on the growth of H. pylori, and this study may be the first to reveal its obviously inhibitory effect on HtrA protein. Our findings imply that Fisetin could be a potential candidate for further research as a therapeutic agent targeting protein HtrA, providing a new direction for the exploration of lead compounds and potential drugs against H. pylori infections.