We examined the effects of the following seven experimental diets that varied in the concentration of inactivated cells of Enterococcus faecalis (Ef) and mannan oligosaccharides (MOS), on Rainbow Trout Oncorhynchus mykiss: control (C) diet (no Ef and no MOS), diet E0.25% (2.5 g/kg Ef), diet E0.5% (5 g/kg Ef), diet M0.25% (2.5 g/kg MOS), diet M0.5% (5 g/kg MOS), diet EM0.25% (2.5 g/kg Ef + 2.5 g/kg MOS) and diet EM0.5% (5 g/kg Ef + 5 g/kg MOS). Rainbow Trout, initially weighing 36.27 ± 0.42 g (mean ± SD) were distributed into fourteen 60‐L glass tanks at a stocking density of 35 fish per tank. Each diet was hand‐fed to duplicate groups of fish twice daily for a 12‐week period. After the feeding test, an intraperitoneal injection challenge test of Aeromonas salmonicida was conducted over 14 d. Resulting data were submitted to a multivariate analysis of variance. Weight gain increased significantly (P < 0.05) in E0.25%, M0.25%, and EM0.5% experimental groups compared with the control. Specific growth rate was significantly higher (P < 0.05) in fish fed E0.25%, M0.25%, and EM0.5% diets compared with fish fed the C diet. Feed gain ratio and protein efficiency ratio were significantly improved (P < 0.05) in fish fed the EM0.5% diet compared with fish fed the C diet. Feed intake, protein efficiency ratio, protein retention, and the apparent digestibility coefficient recorded slight differences within experimental groups. Hematocrit value and phagocytic activity were significantly higher (P < 0.05) in fish fed E0.25%, E0.5%, M0.5%, EM0.25%, and EM0.5% diets compared with fish fed the C diet. Except for fish in the E0.25% group, fish in all other experimental groups showed a significantly higher (P < 0.05) mucus weight compared with those in the C group. After the challenge test, cumulative mortality and frequency of A. salmonicida were significantly decreased (P < 0.05) in all experimental groups compared with the C group. In conclusion, dosage and single or combined supplementation of Ef and MOS are factors that significantly affect fish performance.