Listeria monocytogenesis an important food-borne pathogen whose ability to form disinfectant-tolerant biofilms on a variety of surfaces presents a food safety challenge for manufacturers of ready-to-eat products. We developed here a high-throughput biofilm assay forL. monocytogenesand, as a proof of principle, used it to screen an 80-compound protein kinase inhibitor library to identify molecules that perturb biofilm development. The screen yielded molecules toxic to multiple strains ofListeriaat micromolar concentrations, as well as molecules that decreased (≤50% of vehicle control) or increased (≥200%) biofilm formation in a dose-dependent manner without affecting planktonic cell density. Toxic molecules—including the protein kinase C antagonist sphingosine—had antibiofilm activity at sub-MIC concentrations. Structure-activity studies of the biofilm inhibitory compound palmitoyl-d,l-carnitine showed that whileListeriabiofilm formation was inhibited with a 50% inhibitory concentration of 5.85 ± 0.24 μM,d,l-carnitine had no effect, whereas palmitic acid had stimulatory effects. Saturated fatty acids between C9:0and C14:0wereListeriabiofilm inhibitors, whereas fatty acids of C16:0or longer were stimulators, showing chain length specificity.De novo-synthesized short-chain acyl carnitines were less effective biofilm inhibitors than the palmitoyl forms. These molecules, whose activities against bacteria have not been previously established, are both useful probes ofL. monocytogenesbiology and promising leads for the further development of antibiofilm strategies.