This study investigates the effect of Sargassum ilicifolium algae fermentation using Lactobacillus plantarum and Saccharomyces cerevisiae probiotics on growth, immunity, gene expression, and digestive and antioxidant enzymes before and after challenge with Streptococcus iniae in trout. Trout (50.69 ± 1.33 g) were fed with different percentages of S. ilicifolium (SI) powder over an 8-week trial. The supplements included both unfermented (SI 0.5%, SI 1%, SI 2%) and fermented (FSI 0.5%, FSI 1%, FSI 2%) forms. A significant increase in white blood cells was observed in the group receiving 2% FSI compared to the control group (P <
0.05). All growth factors in trout notably increased with varying levels of fermented and unfermented S. ilicifolium powder compared to the control group (P <
0.05). Biochemical parameters including serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase, and alkaline phosphatase exhibited a notable rise before the challenge with S. iniae (P <
0.05). Immune parameters, including total immunoglobulin, lysozyme, complement 3, bactericidal activity, and nitroblue tetrazolium showed increases both before and after the challenge with S. iniae (P <
0.05). Additionally, the expression levels of the insulin-like growth factor-1, interleukin-6, tumor necrosis factor-alpha, and Ghrelin genes showed significant increases both before and after exposure (P <
0.05). Also, antioxidant enzymes and digestive enzymes increased significantly before and after exposure (P <
0.05). All levels of unfermented and fermented, except for SI 0.5%, showed significant increases compared to the control group following the challenge with S. iniae (P <
0.05). Overall, fermented and unfermented S. ilicifolium support growth and enhance immune, antioxidant, and digestive functions in rainbow trout effectively.