Listeria monocytogenes (LM) is an opportunistic foodborne pathogen responsible for listeriosis in both humans and animals. The disease manifests in a variety of ways, including febrile gastroenteritis, septicemia, meningitis, and in some cases, preterm birth and spontaneous abortion. It is therefore crucial to develop effective strategies to control this bacterium. In this study, we isolated and characterized a novel Listeria phage, named LMLPA3. Morphological and genomic analyses revealed that phage LMLPA3 belongs to the class Caudovirales, family Herelleviridae, Myovirus-like. Phage LMLPA3 demonstrated remarkable stability across a range of pH values (4-10), temperatures (4-50 °C), and high NaCl concentrations (12% w/v). A total of 68 strains, comprising nine serotypes of L. monocytogenes and five other Listeria species, were found to be susceptible to lysis by phage LMLPA3. It is noteworthy that treatment with phage LMLPA3 resulted in a significant disruption of the biofilms formed by seven different serotype strains of L. monocytogenes, in comparison to the control. Furthermore, phage LMLPA3 effectively reduced the number of L. monocytogenes cells by 4 log