This study aimed to isolate and identify Aeromonas hydrophila and to test the pathogenicity of this bacterium and the infectious spleen and kidney necrosis virus (ISKNV) in the Nile tilapia. The single infection experiments aimed to determine the LD50 of A. hydrophila and ISKNV infections. The infections were conducted in two stages: a single infection with A. hydrophila and another with ISKNV, each at different doses. Co-infection doses of A. hydrophila and ISKNV were derived from the LD50 of the single infections. Infections with either A. hydrophila or ISKNV resulted in mortality rates exceeding 50% in each treatment group. The treatments included the A. hydrophila control (K) and IB7, as well as the ISKNV control (K) and P0. The third treatment was co-infection with A. hydrophila and ISKNV (K and P0+IB7). All treatments were observed for 14 days. The results showed that single infections with A. hydrophila, ISKNV, and co-infections produced significantly different outcomes among treatments (P< 0.05). In the A. hydrophila infection, the survival rate of the Nile tilapia in the IB7 treatment was 33%, while it was 100% in the control (K). In the ISKNV infection, the survival rate in the P0 treatment was 20% compared to 90% in the control (K). In the co-infection of A. hydrophila and ISKNV, the survival rate in the P0+IB7 treatment was 10% compared to 90% in the control (K). This study concluded that co-infection with A. hydrophila and ISKNV increases and accelerates mortality compared to single infections with either A. hydrophila or ISKNV. Additionally, hematological immune responses showed significant differences in co-infections compared to single infections.