Inhibitory activity of reuterin (β-hydroxypropionaldehyde) combined with the antimicrobial peptide nisin or the lactoperoxidase system (LPOS) against food-borne Gram-negative pathogens in milk refrigerated at 4 and 8 °C was investigated. At 4 °C, reuterin (8 AU/ml) and LPOS were bactericidal against Salmonella enterica, Campylobacter jejuni, Aeromonas hydrophila and Yersinia enterocolitica, whereas the only effect recorded for nisin was a slight inhibition of Escherichia coli O157H7. At 8 °C, reuterin was bactericidal against all the Gram-negative pathogens studied. Same results were attained with LPOS, except for its effect on Y. enterocolitica which was only bacteriostatic. The combination of reuterin with nisin did not enhance the antimicrobial effect of reuterin. A strong synergistic bactericidal activity of reuterin in combination with LPOS on E. coli O157H7 and S. enterica was observed in milk at 4 °C, and against all the Gram-negative bacteria assayed in milk refrigerated at 8 °C. The application of both antimicrobials would be a useful means to inhibit pathogenic microorganisms, which may be present in milk due to postpasteurization contamination. © 2007 Springer-Verlag.