Extracellular vesicles (EVs) in bacteria have been implicated in invasive and, through enzymes, infective processes. One Gram-positive bacterium lacking any EV research, despite having commercial impacts on the aquaculture industry, is Renibacterium salmoninarum. We addressed this gap in knowledge by utilizing scanning electron microscopy to provide the first reported evidence for the production of EVs by R. salmoninarum strain H-2. Dispersive light scattering detected that the EVs were heterogeneous in size, and the protein compositions were similar to the bacterial membrane and contained the virulent protein factors p22 and p57. The EVs additionally had a concentrated negative charge compared with R. salmoninarum H-2, as determined by Z potential. Finally, these particles seemed to play a role in host invasion in vitro in the salmon head kidney cell line, as demonstrated by the occurrence of a cytotoxic effect within the first 48 hr post-infection. Higher EV concentrations (i.e. 52.6 mu g/ml) were more toxic than R. salmoninarum H-2. This information serves as a foundation to develop and test possible uses for R. salmoninarum EVs in salmon aquaculture, inspiring future advances against bacterial kidney disease.