Food-borne pathogens are an important challenge for the food industry. In this study, the possibility of using the previously designed and synthesized antimicrobial peptide HX-12C as a new food antimicrobial was evaluated. Bacteriostatic and bactericidal tests showed that HX-12C has strong, rapid and broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria. Outer- and inner-membrane permeabilization assays revealed that HX-12C killed food-borne pathogens by inducing membrane permeability. Further Scanning Electron Microscope (SEM) and Transmission Electron Microscope (TEM) also showed that HX-12C can distinctively act on the bacterial membrane leading to the leakage of cellular contents. Moreover, HX-12C also showed anti-biofilm ability in bacterial killing tests. In the food storage test, HX-12C exhibited significant antimicrobial function in raw pork and orange juice. Therefore, HX-12C has shown great potential as a new antimicrobial agent in food storage.