Salmonella spp. are recognised as one of the top four global causes of diarrhoeal disease, with many infections originating from both omnivorous and plant-based diets. Notably, some strains of Salmonella have now evolved to be resistant to multiple antibiotics, presenting a growing challenge to public health. To cause infection, Salmonella must overcome colonisation resistance, a natural defence mechanism whereby the host’s intestinal microbiota prevent invading pathogens from establishing themselves and causing disease. Numerous members of the microbiota, including probiotic strains like E. coli Nissle 1917 (EcN), actively defend their niche by producing antagonistic compounds. Previous work has demonstrated reduced expression of Salmonella Pathogenicity Island 1(SPI-1) regulator hilA when grown in EcN cell-free supernatant. To identify genes involved in the regulatory pathways of probiotic E. coli extracellular factors mediating downregulation Salmonella D23580 hilA expression, Transposon Directed Insertion Site Sequencing (TraDIS) screening was performed on the Salmonella hilA luciferase promoter-reporter strain. Isolation of luciferase revertant colonies grown on agar plates infused with EcN cell-free conditioned media, revealed key regulatory genes, including T3SS regulators hilE and hha-tomB plus novel genes racX and trpS2. This highlights further complexity to the tight regulation of Salmonella invasion and infection, revealing new potential therapeutic targets.