Disrupting bacterial quorum sensing (QS) signaling is a promising strategy to combat pathogenic biofilms without the development of antibiotic resistance. Here, we report that food-associated bacteria can interfere with the biofilm formation of a Gram-negative pathogenic bacterium by targeting its AHL (acyl-homoserine lactone) QS system. This was demonstrated by screening metabolic end-products of different lactobacilli and propionibacteria using Gram-negative and biofilm-forming Chromobacterium violaceum as the QS reporter and our anti-QS microscale screening platform with necessary modifications. The method was optimized in terms of the inoculation technique and the concentrations of D-glucose and L-tryptophan, two key factors controlling the synthesis of violacein, a purple pigment indicating the activation of the QS system in C. violaceum. These improvements resulted in ca. 16-times higher violacein yields and enabled revealing anti-QS effects of Lactobacillus acidophilus, Lentilactobacillus kefiri, Lacticaseibacillus rhamnosus and Propionibacterium freudenreichii, including new cheese-associated strains. Our findings also suggest that acetate and propionate excreted by these species are the main factors that interrupt the QS-mediated signaling and subsequent biofilm growth without affecting the cell viability of the C. violaceum reporter. Thus, the present study reports a revised anti-QS screening method to accurately define new bacteria with an ability to combat pathogens in a safe and sustainable way.