The barramundi (Lates calcarifer) is a fish of high economic value, a feature of which has increased its aquaculture. The expansion of aquaculture not only leads to higher profits but raises the risks involved in aquaculture as well. One of them is a bacterial disease. Pathogenicity testing is essential to understand how these bacteria infect their host, which can inform strategies for managing bacterial diseases. Preliminary tests identified two bacteria, which were subjected to molecular DNA identification and sequencing, revealing the presence of Vibrio navarrensis and Acinetobacter lwoffii. The two bacterial strains were then tested on barramundi fingerlings measuring 6- 7cm to determine the LD50 for subsequent pathogenicity assessment. The pathogenicity tests involved seven treatments with three replicates each, including treatment A, which involved the injection of V. navarrensis 107 CFU mL-1; treatment B with the injection of V. navarrensis at 106 CFU mL-1; treatment C with the injection of V. navarrensis 105 CFU mL-1; treatment D with the injection of A. lwoffii at 107 CFU mL-1; treatment E with the injection of A. lwoffii at 106 CFU mL-1; treatment F with the injection of A. lwoffii at 105 CFU mL-1; and a control group injected with phosphate buffered saline (PBS). The LD50 results indicated that V. navarrensis and A. lwoffii bacterial densities were 1,7 x 105 CFU mL-1 dan 2,1 x 105 CFU mL-1. Significant differences were noted in mortality rates, average time to death, and blood parameter profiles across all treatments compared to the control group (P< 0.05). Histopathological examination of the liver and spleen revealed hepatocyte damage, cell inflammation, melanomacrophage centers, and cell necrosis. Test of the intestine showed hemorrhage and hydropic degeneration in the muscular layer. A series of pathogenicity tests confirmed that these two bacteria are responsible for causing bacterial disease in Barramundi culture in North Aceh.