This study investigated the diversity of bacterial community in healthy and streptococcosis infected Nile tilapia (Oreochromis niloticus) to identify the primary causative agents associated with the disease using metagenomics, phylogenetic analysis and in vivo challenge test. A total of 24 fishes, both healthy and diseased were collected during summer from three different districts and six different ponds of Bangladesh. Alpha-beta diversity analysis of the next generation sequence data showed distinctly different (P <.05) microbial communities in control and diseased fishes. In diseased fish, we found significant (P <.05) increase abundance of Enterococcus in the skin lesion, and Leifsonia, Photobacterium, Aeromonas, Pseudomonas in the gut. Interestingly, three different Enterococcus species, E. faecalis, E. hirae and E. faecium were identified the causative agents of streptococcosis through 16S rRNA based phylogenetic analysis. In-vivo challenge test also revealed the high pathogenicity and mortality of these species to experimental tilapia fingerlings. Further study revealed a significant correlation between the pathogenicity and sequence divergence in characterized Enterococcus spp. isolates. Taken together, our study for the first time demonstrated the dominance of multiple Enterococcus species as causative agents of streptococcosis in Nile tilapia in Bangladesh. Our findings should be valuable for the diagnosis and treatment of streptococcosis infection in tilapia.