Mass Galla chinesis et camelliae Fermentata (Chinese gall leaven, CGL) was investigated for activities against Helicobacter pylori (H. pylori) both in vitro and in vivo. The agar dilution method and time-kill curves, as in vitro assays and an in vivo study using a Kunming mice model, were performed. CGL demonstrated a strong anti-Helicobacter pylori activity in vitro with the minimal inhibitory concentrations (MICs) against multiple H. pylori strains of 0.5~8 mg/ml and the decreasing trend time-kill curves when increasing CGL concentrations. H. pylori eradication rates in vivo were evaluated based on rapid urease test (RUT) and histopathologic criteria. Results revealed that the eradication rates in the CGL groups were 40% (4/10) in the high dosage group, 33% (4/11) in the medium dosage group, and 18% (2/11) in the low dosage group, with the difference between the high dosage and H. pylori control groups being significant (P = 0.035). The H. pylori colonization scores could be reduced partly by CGL. These in vivo results demonstrated that CGL in a rationally high dosage might be the most effective.