Listeria monocytogenes is a foodborne pathogen and causes illnesses with a high mortality rate in susceptible populations. Several dairy-related outbreaks have been attributed to contamination by L. monocytogenes, which requires antimicrobial interventions to enhance the safety of these products. This study aimed to determine the antimicrobial activity of the ohelo berry (Vaccinium calycinum), a Hawaiian wild relative of cranberry, against L. monocytogenes in culture media and milk products. The effect of ohelo berry juice at its sub-inhibitory concentrations on the physicochemical properties, biofilm formation, and gene expression of L. monocytogenes was also investigated. The minimum inhibitory concentration of ohelo berry juice against L. monocytogenes was 12.5%. The sub-inhibitory concentration of ohelo berry juice (6.25%) significantly increased the auto-aggregation and decreased the hydrophobicity, swimming motility, swarming motility, and biofilm formation capability of L. monocytogenes. The relative expression of genes for motility (flaA), biofilm formation and disinfectant resistance (sigB), invasion (iap), listeriolysin (hly), and phospholipase (plcA) was significantly downregulated in L. monocytogenes treated by the 6.25% juice. L. monocytogenes was significantly inhibited in whole and skim milk supplemented with 50% ohelo berry juice, regardless of the fat content. These findings highlight the potential of ohelo berry as a natural preservative and functional food to prevent L. monocytogenes infection.