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The anti-microbial properties of acetone extracts from <i>Hibiscus sabdariffa</i> calyces, fractions isolated by silica gel chromatography and hibiscus acid purified from some of these fractions and additionally identified by nuclear magnetic resonance spectroscopy, mid-infrared spectroscopy and X-ray diffraction, were studied against both multidrug-resistant <i>Salmonella</i> strains and pathogenic <i>Escherichia coli</i> bacteria. Gel diffusion was used to determine the anti-microbial effects. The mode of action of hibiscus acid was determined by crystal violet assay. Hibiscus acid and 17 of the 25 chromatographic fractions obtained, displayed an anti-microbial effect against all bacterial strains tested. Hibiscus acid showed a greater anti-microbial effect than the acetone extract against most of the bacteria strains, while chromatographic fractions IX&#8722;XIV exerted the greatest anti-microbial effect against all bacteria. The minimum inhibitory concentration of the acetone extract was 7 mg/mL, and the minimum bactericidal concentration was 10 mg/mL, while the corresponding values for hibiscus acid were 4&#8722;7 and 7 mg/mL, respectively. The results of the crystal violet assay indicate that hibiscus acid alters membrane permeability. Hibiscus acid is a potential alternative to control multidrug-resistant bacteria. Due to its ready availability and easy extraction from <i>H. sabdariffa</i>, hibiscus acid is potentially useful in the food industries.