In this study, we examined the effects of the following eight experimental diets, which varied in fructo oligosaccharides (FOS), mannan oligosaccharides (MOS) and Bacillus clausii concentrations, on the Japanese flounder: control diet (no FOS, MOS and B. clausii), diet F (5 g kg−1 FOS), diet M (5 g kg−1 MOS), diet FM (2.5 g kg−1 FOS + 2.5 g kg−1 MOS), diet B (107 cells g−1B. clausii), diet FB (5 g kg−1 FOS + 107 cells g−1B. clausii), diet MB (5 g kg−1 MOS + 107 cells g−1B. clausii) and diet FMB (2.5 g kg−1 FOS + 2.5 g kg−1 MOS + 107 cells g−1B. clausii). Japanese flounder, initially weighing an average of 21 g, were distributed into 24 net cages at a stocking density of 20 fish per cage. Each diet was hand‐fed to three groups of fish twice daily for 56 days. The weight gain rate (WGR) in fish fed diets B, MB and FMB were significantly higher than in fish fed the control diet, where the fish fed diet FMB had the highest WGR. Fish fed any of the diets, except diets F and B, exhibited better feed conversion ratio than those fed the control diet. Diets MB and FMB significantly elevated intestinal protease activity compared with the control diet, but only the diet FMB promoted amylase activity. Feeding diets FB and FMB increased body protein deposition; additionally, feeding diets B, MB and FMB significantly reduced body lipid deposition. Lysozyme (LSZ) activity was significantly higher in fish fed diets B, FB, MB and FMB than in fish fed the control diet. All diets, except diet M, decreased triglyceride (TG) levels compared to the control diet. Low‐density lipoprotein cholesterol levels in fish fed diets F, FB and FMB were significantly lower than in fish fed the control diet. Without exception, no diets affected feeding rate, condition factor, body moisture, ash contents, phagocytic activity of leucocytes or cholesterol or high‐density lipoprotein cholesterol levels. Our results suggest that diets supplemented with FOS, MOS and B. clausii improved growth performance and health benefits of the Japanese flounder more than other diets or the control diet.