Strain Endo-EH was isolated from a root-filled tooth associated with periradicular lesions. After subculturing on Columbia blood agar, phenotypic and genomic characterizations using different biochemical test systems, automated ribotyping, MALDI-TOF mass spectronomy, antibiotic susceptibility testing, and 16S rRNA gene sequence analysis were applied for further analysis. Phenotypic characterization identified this strain as Vagococcus fluvialis. Riboprint pattern analysis and 16S rRNA sequencing clearly separated it from relevant genera such as Enterococcus and Tetragenococcus and also from other Vagococcus species. This taxon is a new entry to the list of more than 200 microbial species detected in infected root canal systems.