The misuse of antibiotics has led to high levels of drug-resistance in specific pathogens, promoting the search of molecules from different natural sources or the design of novel drug candidates. Medicinal and edible plants are a rich source of bioactive compounds, in particular those of polyphenol class. In the present work, we screen the antimicrobial properties of an aqueous extract prepared from the leaves of Ugni molinae (Turz) against a panel of pathogenic bacteria strains formed by Helicobacter pylori (ATCC 43504), Listeria monocytogenes (ATCC 7644), Staphylococcus aureus (ATCC 9144), Escherichia coli (ATCC 11775), and Salmonella enterica (ATCC 13076). Preliminary fast HPLC-micro fractionation allows the identification of potential urease inhibitors using high throughput urea-phenol microplate assay. Afterwards, preparative fractionation by Centrifugal Partition Chromatography (CPC) allow to select the specific bioactive fractions. A combination of antimicrobial tests, enzyme assays and molecular docking resulted in the identification by HPLC-MS/MS of two quercetin-O-(6´´-O-galloyl)-hexosides as the most dominant compounds in the active CPC-fractions. These bioactive compounds were quercetin-3-O-(6´´-O-galloyl)-b-galactopyranoside (hyperin 6”-gallate) and quercetin-3-O- b -D-(6´´-O-galloyl)- b -glucopyranoside (tellimoside). The molecular docking evaluation revealed that hyperin-6´´-gallate enter the binding site of urease and bind in through pi-cation, pi–pi, and H-bond interactions. In concordance with the in-silico assay, the CPC fraction containing this compound has the lowest values of IC50 for Jack bean (0.41 ± 0.08 µg/mL) and Helicobacter pylori (0.28 ± 0.08 µg/mL) ureases, respectively. Moreover, Label-Free Microscale Thermophoresis (MST) analysis suggest that this flavonoid forms a complex with urease, even inducing protein aggregation. In conclusion, Ugni molinae leaves has potent anti-urease flavonoids that can contribute to significantly reduce the acclimation of H. pylori in the acidic environmental of gastric mucosa.