Enterococci are members of the natural microbiota of animal and human intestinal tracts and are capable of causing opportunistic infections. They are also used as starter cultures in the food industry as well as in health supplements and probiotics by the pharmaceutical industry. This Janus-faced status requires a careful evaluation on the basis of pathogenic traits to ensure the safety of the strain used to produce food and pharmaceuticals. We performed gapped-genome sequencing of a probiotic strain Enterococcus faecalis Symbioflor 1 and present initial results deriving from comparative genome analysis with that of the previously sequenced pathogenic clinical isolate E. faecalis V583. There was strong overall conservation of synteny between both strains and a detailed analysis revealed the absence of large genomic regions from the chromosome of the probiotic strain, indicating gene loss. Genes absent from the Symbioflor 1 strain included those encoding the enterococcal cytolysin, enterococcal surface protein, and gelatinase (coccolysin) as well as hyaluronidase and the peptide antibiotic AS-48. This data was confirmed using PCR primers specific for the respective genes. However, other enterococcal determinants such as aggregation substance, collagen adhesion protein, the ability to resist oxygen anions as well as capsule formation were detected. The presence of these traits may be advantageous for the strain Symbioflor 1 since they potentially enable colonization and proliferation of the bacterium on mucosal surfaces thereby conferring on it probiotic traits.