Salmonella enterica serovar Enteritidis (S. Enteritidis) is the most common causative agent of non-typhoidal salmonellosis in Malaysia. We aimed to characterize S. Enteritidis isolated from humans and animals by analyzing their antimicrobial resistance profiles and genotypes. A total of 111 strains were characterized using multiple-locus variable-number tandem repeat analysis, pulsed-field gel electrophoresis, and antimicrobial susceptibility testing. Both typing methods revealed that genetically similar S. Enteritidis strains had persisted among human and animal populations within the period of study (2003-2008). Only 39% of the strains were multi-drug resistant (i.e., resistant to 3 or more classes of antimicrobial agents), with a majority (73%) of these in low-risk phase (multiple antibiotic resistant index <0.20). Limited genetic diversity among clinical and zoonotic S. Enteritidis suggested that animals are possible sources of human salmonellosis. The degree of multi-drug resistance among the strains was generally low during the study period.