Objective <p>Probiotic bacteria can inhibit the growth of harmful bacterial species but may be limited in the bacteria they can counteract. We developed a novel co-culture assay in which candidate probiotic bacteria can be co-incubated with both Gram-positive and Gram-negative reporter pathogens that have been modified to emit light, so as to monitor pathogen growth simply by quantitation of emitted light from the culture. We used this assay to identify a novel probiotic bacterium with anti-microbial activity against both Gram-positive and Gram-negative pathogens.</p> Results <p>We isolated a novel bacterium from non-pasteurized milk and identified it as a strain of <i>Lactobacillus delbrueckii</i>. Testing in our assay confirmed that this bacterium was able to inhibit the growth of both <i>Staphylococcus aureus</i> and <i>Escherichia coli</i>, but in a delayed fashion, after allowing a period of early growth. This activity was not dependent on living cells but also a property of conditioned medium. We posited that this was because the pathogens stimulated the production of an inhibitory factor from the<i> Lactobacillus</i>, presumably a bacteriocin peptide. Pre-treatment of conditioned medium with a protease abolished this inhibitory activity, indicating that <i>L. delbrueckii</i> is stimulated to produce an antibacterial peptide in the presence of a bacterial pathogen.</p>