Considerable challenges remain in the development and application of natural antimicrobial substances for food preservation to meet food safety needs. In this paper, ehimensins F4, F5, and F6, three antimicrobial peptides produced by Paenibacillus ehimensis HD, were purified by using macroporous adsorption resin and RP-HPLC. The molecular mass of ehimensins F4 and F5 was 1101 Da and that of ehimensin F6 was 1115 Da. LC-MS/MS demonstrated that each ehimensin consisted of fatty acids, 2,4-diaminobutyric acid, and amino acids. Mean-while, circular dichroism revealed that each ehimensin was mainly composed of random coil and & beta;-sheet structures. The three ehimensins maintained high activities under different conditions because they were stable over a wide range of temperatures and pH and in the presence of protease. Moreover, they exhibited low he-molysis activities and obvious antimicrobial activities against various food pathogenic bacteria and fungi. Soaking the strawberries in 1.0 mg/mL ehimensins for 1 min at room temperature could significantly inhibit fungal growth. And the shelf life of the strawberries was extended from 1 day to 4 days. These results suggested that ehimensins from P. ehimensis HD have great potential for use as biopreservatives to improve food safety.