Nile tilapia (Oreochromis niloticus) and tambaqui (Colossoma macropomum) are the two most produced freshwater fishes in Brazil. This study investigated the potential pathogenicity of Streptococcus agalactiae and Francisella orientalis, previously isolated from diseased Nile tilapia, to tambaqui. Experimental infection trials were conducted in juvenile tambaqui at a dose of approximately 107 CFU fish−1, assessing clinical signs, mortality, bacterial recovery, and histopathological changes. Results demonstrated that S. agalactiae exhibited high pathogenicity to tambaqui, causing rapid disease progression, high mortality (83.33%) within 48 h post-infection, and severe lesions in multiple organs, under the experimental conditions. In contrast, F. orientalis infection did not result in mortality or clinical signs, despite bacterial recovery and granulomatous inflammation observed in the tissues. This study highlights the need to consider the potential impact of these pathogens in tambaqui farming.