This study was conducted to identify the biological characteristics of pathogen responsible for the death of Tibetan sheep in Nagarze County, Tibet, China. For this purpose, samples were collected from diseased animals and for bacterial culture and isolation. The isolated strains were subjected to several tests which included gram in-vitro drug sensitivity and in-vivo pathogenicity tests. The results revealed that 7 strains of gram-positive cocci were isolated from Tibetan sheep, named TS-1, TS-2, TS-3, TS-4, TS-5, TS-6, and TS-7. These strains exhibited specific biochemical characteristics consistent with Enterococcus faecalis. Whereas, PCR amplification results were consistent with the expected outcomes on target band of approximately 1500 bp. Genetic evolutionary analysis revealed a significant homology (96.0%99.9%) between the isolates and Enterococcus faecium. In-vitro drug sensitivity tests demonstrated that all the isolates exhibiting multiple drug resistance. Furthermore, the isolated strains displayed varying degrees of pathogenicity in mice. This study confirms that Enterococcus faecium is the causative agent for the deaths of Tibetan sheep. These findings enhance our understanding of the disease and suggest valuable insights for its prevention, control, and future research.