This study evaluated the pathogenicity of Aeromonas veronii strain BLB-01 (GenBank: MF370515) in red-bellied pacu (Piaractus brachypomus), an emerging freshwater species with increasing farming potential in India. P. brachypomus Fingerlings (10±2 g) were intraperitoneally challenged with graded doses (105–109 CFU/mL) A. veronii under controlled laboratory conditions following OECD guideline 203. Probit analysis (Finney 1952) estimated the median lethal dose (LD50) at 7.8 × 106 CFU mL−1, confirming the virulence of the isolate. Clinical signs, mortality, and haematological (RBC, WBC, haemoglobin) and serum biochemical markers (glucose, albumin ALT, AST, ALP) were monitored. Infected fish displayed motile Aeromonas septicemia (MAS) signs: erratic swimming, mucus hypersecretion, skin haemorrhages, and periorbital erythema, 7 days post-infection. Haematological analysis revealed decreased haemoglobin and total erythrocyte count (TEC) indicating anaemia, increased total leukocyte count (TLC) signifying leukocytosis, reduced platelets denoting thrombocytopenia, and macrocytic-hypochromic shifts (elevated MCV and MCH; reduced MCHC; p<0.05). Serum biochemistry showed increased glucose levels reflecting stress hyperglycaemia, elevated Alanine aminotransferase (ALP), Aspartate aminotransferase (ALT), and Alkaline phosphatase(AST) indicating hepatic injury, and decreased albumin signifying hypoalbuminemia (p<0.05). Histopathology confirmed severe gill lamellar fusion and hyperplasia, hepatic pyknosis, karyorrhexis and vacuolation, plus renal haemorrhage with melanomacrophage aggregates. This study highlights the vulnerability of P. brachypomus to Aeromonas infections and provides critical LD50 data for risk assessment in aquaculture systems.These multisystemic disruptions underscore Aeromonas veronii high virulence in Piaractus brachypomus, necessitating stringent biosecurity, early diagnostics, and immunoprophylaxis strategies for sustainable aquaculture farming.