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The present study explored the effect of quercetin on the expression of virulence genes actA, inlA, inlC, and their regulatory components, sigB and prfA, in L. monocytogenes. Furthermore, the physicochemical changes on the surface, membrane permeability, and biofilm formation of quercetin-treated bacteria were evaluated. An inhibitory dose-dependent effect of quercetin (0.1–0.8 mM) was observed on the cell attachment on stainless steel at 2 and 6 h at 37 °C. Quercetin at 0.8 mM prevented the biofilm formation on stainless steel surfaces after 6 h of incubation at 37 °C, while the untreated bacteria formed biofilms with a cell density of 5.1 Log CFU/cm². The microscopic analysis evidenced that quercetin at 0.2 mM decreased the biovolume and covered area of the attached micro-colonies. Also, sigB, prfA, inlA, inlC, and actA genes were downregulated by 7–29 times lower compared to untreated bacteria. In …