The prevalence ofListeria monocytogenesin bovine bulk-tank milk (BTM) in Greece has not been previously investigated. The aim of the study was to estimate the prevalence ofL. monocytogenesin bovine BTM in Greece and to characterize the isolates in terms of carriage of genes encoding for pathogenic determinants, assess the isolates’ biofilm-forming ability and determine their susceptibility against 12 antimicrobials. Samples (n = 138) of bovine BTM were obtained from farms located throughout Northern Greece and were analyzed qualitatively and quantitatively forL. monocytogenes. Five samples (3.6%) tested positive forL. monocytogenes. The pathogen’s populations in these positive samples were below 5 CFU/mL. Most isolates belonged to the molecular serogroup “1/2a, 3a”. All isolates carried the virulence genesinlA,inlC,inlJ,iap,plcAandhlyA, butactAwas detected in only three isolates. The isolates displayed weak to moderate biofilm-forming ability and distinct antimicrobial resistance profiles. All isolates were characterized as multidrug resistant, with resistance to penicillin and clindamycin being a common feature. Considering thatL. monocytogenesconstitutes a serious public health threat, the key findings of the study, related to the carriage of virulence genes and multidrug resistance, highlight the importance of continued monitoring of the pathogen in farm animals.