The main cause of non-typhoidal Salmonella (NTS) infection in humans is ingestion of contaminated animal-derived foods such as eggs, poultry and dairy products. These infections highlight the need to develop new preservatives to increase food safety. Antimicrobial peptides (AMPs) have the potential to be further developed as food preservative agents and join nisin, the only AMP currently approved, for use as a preservative in food. Acidocin J1132β, a bacteriocin produced by probiotic Lactobacillus acidophilus, displays no toxicity to humans, however it exhibits only low and narrow-spectrum antimicrobial activity. Accordingly, four peptide derivatives (A5, A6, A9, and A11) were modified from acidocin J1132β by truncation and amino acid substitution. Among them, A11 showed the most antimicrobial activity, especially against S. Typhimurium, as well as a favorable safety profile. It tended to form an α-helix structure upon encountering negatively charged-mimicking environments. A11 caused transient membrane permeabilization and killed bacterial cells through membrane depolarization and/or intracellular interactions with bacterial DNA. A11 maintained most of its inhibitory effects when heated, even when exposed to temperatures up to 100 °C. Notably, it inhibited drug-resistant S. Typhimurium and its monophasic variant strains. Furthermore, the combination of A11 and nisin was synergistic against drug-resistant strains in vitro. Taken together, this study indicated that a novel antimicrobial peptide derivative (A11), modified from acidocin J1132β, has the potential to be a bio-preservative to control S. Typhimurium contamination in the food industry.