S. enterica is an important foodborne pathogen worldwide. As some strains can form biofilms which may offer protection against antimicrobials, it is of interest to explore ways to prevent biofilm formation by S. enterica. In this study, we engineered a short β-sheet peptide WK2 (WKWKCTKSGCKWKW-NH2) and examined its antimicrobial and anti-biofilm activities against various S. enterica strains, including the multidrug-resistant S. Typhimurium DT104. WK2 displayed bacteriostatic activity with a geometric mean (GM) minimum inhibitory concentration (MIC) of 4.17 μg/mL, and bactericidal activity, with a GM lethal concentration (LC) of 7.51 μg/mL. Crystal violet staining and fluorescence measurements demonstrated that WK2 inhibited S. Typhimurium DT104 biofilm formation at 0.5 μg/mL and killed the sessile cells in biofilms at 8 μg/mL. Real-time polymerase chain reaction (qPCR) and microscopic observation revealed that the anti-biofilm activity of WK2 likely arises through the formation of complexes with bacterial DNA, inhibition of surface organelle biosynthesis and interference with autoinducer-2 (AI-2)-mediated quorum sensing (QS). Therefore, WK2 is a promising antimicrobial agent for the prevention and control of biofilms produced by multidrug-resistant S. enterica.