Nile tilapia, Oreochromis niloticus (O. niloticus) of average body weight (24.5±1.6 g) were fed basal diet supplemented with fructooligosaccharide (FOS) at a concentration of 0, 10, 20 and 30 g/kg diet for 49 days (42 days before and 7days post-challenge). At 42 days of feeding, fish of all groups were challenged intra-peritoneal with 0.2 ml Aeromonas hydrophila (A. hydrophila) (1.5×108 CFU ml-1). Serum and tissue samples were collected on eighth day post infection. Results showed that cortisol level, liver enzymes (alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) and urea level were significantly decreased (P < 0.05) with dietary FOS in comparison with infected group fed basal diet. Furthermore, malondialdehyde (MDA) level as well as superoxide dismutase (SOD), glutathione peroxidase (GPX) and glucose 6 phosphate dehydrogenase (G6PD) revealed significant decrease in the treated groups fed 20 g dietary FOS over the infected group fed basal diet. Moreover, total protein and globulin level as immune component were significantly increased (P < 0.05) in the group treated with 20 g dietary FOS. Feeding O. niloticus supplemented diet with FOS increased survivability after challenging with A. hydrophila. Histopathological examination of infected O. niloticus revealed various pathological alterations in spleen, liver and kidneys represented mainly in hemorrhage, necrosis, with inflammatory reaction. The severity of these changes was reduced in the infected fish groups fed different dietary concentrations of FOS with variable degrees especially at both concentrations of (20g and 30g FOS/ kg diet). In conclusion, these findings indicated that dietary supplementation of FOS has a protective role in challenged O. niloticus with A. hydrophila and could minimize the physiological alterations, increase host immune system, and relieve oxidative stress as well as renewal of tissue histological structures.