Background: Artemisia herba alba, classified into the family of Asteraceae, is an aromatic herb that is traditionally used as a purgative and antipyretic folk medicine by rural people of south Tunisia. This study reports the first identification of antimicrobial peptides from this medicinal plant that inhibited the growth of several food-borne pathogenic bacteria. Materials and methods: The extraction and purification of peptidic agents from Artemisia herba alba, have been performed using precipitation by ammonium sulfate of a phosphate buffer crude extract obtained from the plant leaves, followed by reverse-phase HPLC on a C18 column. The mass of the peptides was estimated by SDS-PAGE electrophoresis, followed by a gel overlay assay and ultra-filtration through a 5 kDa cut-off membrane. Fractions from every purification steps were sampled and assayed for activity towards different food-borne bacterial strains pathogenic and non pathogenic to humans. Results: The phosphate buffer crude extract, as well as its ammonium sulfate precipitate, designated AS-P, inhibited the growth of Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus sensu stricto and the new approved species Bacillus cytotoxicus. AS-P MICs (minimum inhibitory concentrations) ranged from 0.241 to 3.8 mg/ml proteins for L. monocytogenes and B. cereus sensu stricto (strains ATCC10987 and IP5832), respectively. The bioactive AS-P molecules were stable up to 10 minutes heating at 120 degrees C and they resisted organic solvent effects. Antimicrobial activity of A. herba alba AS-P decreased to 40 and 60% after proteolytic treatment with trypsin and proteinase K, respectively, suggesting peptides being responsible for the A. herba alba AS-P activity. The mass of antibacterial A. herba alba peptides was estimated below 5 kDa. Two AS-P fractions, eluted at 40 and 37% acetonitrile, showed antibacterial activity when assayed against L. monocytogenes. Conclusion: A. herba alba could make a new source of novel natural anti-infective agents that could be used in food bio-preservation as natural additives or in human infectious disease treatments against multi-drug resistant pathogens.