Listeria monocytogenes is a causative agent for pernicious listeriosis outbreaks worldwide and it has attained more alertness due to the emergence of resistance and mortality rate. A transcriptional regulator (prfA) regulates all the virulence cascades in L. monocytogenes making it a unique and putative target for many drug molecules. Propolis, nature's hidden treasure, is a complex and heterogeneous mixture that contains many secondary metabolites of plant origin. Bees might produce some active compounds and harnessing their bioactive potential is a burgeoning interest to treat various illnesses. In this context, the present study focuses on evaluating the targeted action of the major bioactive compounds of propolis against the putative target (prfA) of L. monocytogenes. Among various propolis compounds, 75 ligands were selected and docked with the A chain of prfA. Initially, the pharmacokinetic properties of the ligands were evaluated using QikProp v_5.8 in the Schrodinger suite 2023_3. All the pharmacokinetic parameters were satisfied with the selected propolis compounds and the docking score of the compounds obtained was in the range of −13.022 to −5.171 kcal/mol. The compounds with high negative docking scores, such as Ligand 70 (−13.022 kcal/mol) and Ligand 39 (−12.58 kcal/mol) were subjected to molecular dynamics simulation studies to determine their binding stability for a 100 ns simulation course using Desmond v_5.6 packages embedded in the Maestro software v_11.8, followed by the binding free energies of the ligand-receptor complexes were computed using prime MM/GBSA. All the parameters have envisaged the stability of the ligand molecules at the active site of prfA (target protein) to inhibit L. monocytogenes pathogenicity in the host. In sum, the compounds of propolis synergistically act against L. monocytogenes by actively inhibiting the activity of transcriptional regulators (prfA) to combat listeriosis outbreaks, thus ensuring food safety and public health.