Aims The aim of this study was to evaluate the genetic diversity and resistance phenotypes of Listeria monocytogenes strains isolated from clinical encephalitis cases, and compare this population to isolates derived from tank milk of healthy animals. Methods and Results A total of 57 L. monocytogenes strains isolated from ruminant's listeriosis cases (n = 31) and from tank milk of healthy ruminants (n = 26) were characterized by species PCR, molecular serotyping, PCR detection of virulence genes, pulsed-field gel electrophoresis and antimicrobial susceptibility testing. All strains possessed inlA, inlC, inlJ, plcA, actA, hlyA and iap virulence-associated genes while serotyping analysis revealed that they were mainly assigned into IVb group. Genotyping revealed 50 pulsotypes among the 57 strains assigned into seven clusters while indistinguishable pulsotypes between clinical and milk strains were not identified. Resistance of L. monocytogenes isolates to 14-16 antimicrobial agents tested was observed and 23 antimicrobial resistance profiles (ARPs) were defined while no apparent predominant ARP type was observed among isolates. Conclusions Small ruminants are exposed to a broad range of antimicrobial-resistant as well as genetically diverse strains of L. monocytogenes carrying virulence-associated genes but not all of them associated with the disease. Pulsed-field gel electrophoresis analysis suggests that pulsotypes associated with encephalitis are found in farms only in association with listeriosis. Significance and Impact of the Study These findings are valuable in understanding the ecology of this important food-borne pathogen and creating awareness for the emerging antimicrobial resistance.