Persister cells are a sub-population of growth-arrested bacteria with the ability to transiently tolerate bactericidal levels of antimicrobial agents. Listeria monocytogenes is known to form persister cells upon exposure to high concentrations of these agents and later restore to an active, growing state under favorable conditions. This capability is thought to be a key factor in the survival of foodborne pathogens, such as L. monocytogenes, in food and feed production environments. Therefore, this study aimed to investigate the formation of persister cells in two L. monocytogenes strains isolated from a dairy food production environment. Both strains were exposed to high concentrations of gentamicin and nisin for 2, 4, and 6 hours. The results revealed a biphasic killing curve for both strains, a fundamental characteristic of persister cells. According to our results, a reduction of at least 2 Log(CFU/mL) was observed under most tested conditions. After 6 hours, all conditions stabilized at 8 Log(CFU/mL) or below. These findings demonstrate that L. monocytogenes persister cells can be successfully isolated upon exposure to both gentamicin and nisin. This methodology can potentially be used to isolate and further study L. monocytogenes persister cells, contributing to a better understanding of the adaptive phenotypic traits that may enable certain strains to persist in industrial settings.