Abstract <p>This study investigated the effects of nisin concentrations on the biofilm formation and cellular invasion of <i>Listeria monocytogenes</i>. The study also examined the toxicity of nisin concentrations on human cell lines. Nisin is a lantibiotic with antimicrobial properties. <i>L. monocytogenes</i> is a deadly foodborne pathogen leading to listeriosis. Biofilm formation, cell invasion, toxin-antitoxin systems, and extracellular proteins are the primary virulence mechanisms of <i>L. monocytogenes</i>. The PCR method was used to confirm the presence of virulence genes in <i>L. monocytogenes</i>. A microplate assay was used for biofilm quantification. The effect of nisin on cell invasion and cytotoxicity in the HT-29 human cell line was investigated using invasion assay and thiazolyl blue tetrazolium bromide (MTT) methods, respectively. Nisin solutions of 10⁴, 10³, and 10² IU/mL inhibited biofilm formation by 56, 90, and 99%, respectively (<i>p</i> &lt; 0.0001). Nisin at concentration of 10² IU/mL reduced the expression of biofilm formation genes: 2.5-fold for <i>mazF</i>, 3.9-fold for <i>flaA</i>, and 3.4-fold for <i>cheA</i>. However, the increased <i>iap</i> gene expression (<i>p</i> &lt; 0.0001) observed at nisin of 10² IU/mL. Nisin solutions of 10⁴, 10³, and 10² IU/mL reduced the invasion into HT-29 cells by 42, 73, and 99%, respectively. Survival of the HT-29 cells treated with nisin solutions of 10⁴, 10³, and 10² IU/mL decreased by 2, 7, and 18%, respectively. The action of nisin can be enhanced through various innovative techniques, increasing its effectiveness against <i>L. monocytogenes</i>. Nisin significantly inhibited the expression of genes involved in biofilm formation, cell invasion, and cellular signal transduction in <i>L. monocytogenes</i>.</p>