Helicobacter pylori (H. pylori) is the leading cause of infectious gastritis, and its resistance to antibiotics is becoming increasingly significant. Probiotics have been widely reported to have antagonistic effects on H. pylori. In this study, 52 strains of lactic acid bacteria (LAB) were isolated from the feces of healthy human and animal. The acid tolerance, anti-H. pylori ability, urease inhibition rate, and nickel adsorption rate of 19 LAB were determined. PCA evaluation revealed Lactiplantibacillus plantarum (L. plantarum) NCUA001057, Pediococcus pentosaceus (P. pentosaceus) NCUH006018, and L. plantarum NCUH001090 displaying the best comprehensive performance. Subsequently, the hemolytic, bile salt tolerance, antioxidant capacity, co-aggregation ability with H. pylori, and adhesion rate to AGS cells of three LAB were evaluated. Moreover, three LAB downregulated the production of IL-1β and IL-8 in H. pylori pre-treated AGS cells. We preliminarily studied the antibacterial substances of three LAB which mainly included acid, hydrogen peroxide, and protein, and the antibacterial substances are heat-tolerant. Furthermore, we found that the main modes of antibacterial action of P. pentosaceus NCUH006018 were as follows: inhibited the adhesion of H. pylori to AGS cells, made H. pylori spherical and destroyed its intact cell structure, and downregulated the metabolism genes of H. pylori flagellin and ribosome. In this study, the high potential of antagonizing H. pylori by three novel LAB was demonstrated, and their mode of antibacterial action provided a new strategy for prevention or inhibition of H. pylori infection.