Introduction: Salmonella Livingstone is one of the most common serotypes responsible for nosocomial outbreaks in Tunisia. In this study, 42 isolates of Salmonella Livingstone were analyzed. Most of these were isolated from humans (31 strains from Tunisia and 9 strains from Belgium) and 2 isolates came from food products (beef and pork). Methodology: All strains were characterized by antibiogram, multilocus sequence typing (MLST), and virulotyping. This last technique was carried out by simple PCR of five chromosomal genes (agfA, hin/H2, iroB, phoP/Q, and slyA) and two plasmid genes (spvA and spvC). Results: All Tunisian strains were resistant to amoxicillin, amoxicillin-clavulanic acid, ticarcillin, cefalotin, gentamicin, and kanamycin. They were also resistant to third-generation cephalosporin antibiotics (cefotaxim and ceftazidim). Belgian isolates were susceptible to all antibiotics tested. Further to MLST analyses, Tunisian strains belonged to the same sequence type, ST543. For Belgian isolates, eight strains had a ST543 profile, two strains had a ST638 profile, and one strain had a ST457 profile. Analyses of the virulence gene contents showed that strains isolated in different years and from different origins had the same virulence profile. These carried all five chromosomal genes and lacked plasmid-located virulence genes spvA and spvC. Conclusions: A combination of different typing methods showed that the majority of Belgian strains and all Tunisian strains were closely related; they belonged to the same sequence type (ST543) and had the same virulence profile, but different antibiotic resistance profiles depended on the country of origin.