One of the major challenges threatening the long-term sustainability of aquaculture is the impact of sudden outbreaks of pathogenic microorganisms, whether endemic or introduced, with sanitary importance. Flavobacterium psychrophilum is the causative agent of flavobacteriosis, which affects intensive salmonid production systems worldwide. The most common strategies for controlling this pathogen are antibiotics and vaccines. However, these measures may prom ote bacterial resistance, do not guarantee effective immunization against diverse strains, and are economically costly. Consequently, their use is now increasingly restricted and tightly regulated globally. In this context, the incorporation of plant-based substrates biotransformed by the mycelia of edible fungi into the diet of farmed fish emerges as a cost-effective and environmentally friendly immunostimulant alternative. In this work, we evaluated whether the inclusion of 10% wheat flour biotransformed by Pleurotus ostreatus mycelium (over 21 days) in the diet of Oncorhynchus mykiss juveniles enhances parameters of the innate immune response following exposure to sublethal concentrations of a virulent F. psychrophilum strain. This strain was isolated from diseased fish at the CEAN facilities, identified and characterized based on its virulence through biochemical and microbiological methods, and confirmed by non-quantitative PCR. For the feeding trial, 64 juvenile trout were distributed into 16 tan ks (50 L each) in an open-flow system supplied with water from the Chimehuín River. Four experimental groups were established (n = 4 tanks per group): Control: Base diet (BD), injected with PBS; Biotransformed: Enriched diet (ED), injected with PBS; Contro l-Flavobacteria: BD injected with 10⁶ CFU/fish of F. psychrophilum ; Biotransformed -Flavobacteria: ED injected with 10⁶ CFU/fish of F. psychrophilum. BD and ED were formulated according to the nutritional requirements of rainbow trout and subsequently characterized for their proximate composition. All groups underwent a 7-day acclimation to their respective diets. The feeding period lasted 21 days, followed by 10 days post-injection. Fish were anesthetized for whole blood collection and then euthanized according to an approved protocol. Phagocytic capacity was measured in whole blood, while lysozyme, peroxidase, and antiprotease activities were assessed in serum. Data were analyzed using two-way ANOVA. Results for phagocytosis and antiprotease activity showed no combined effect of diet and bacterial challenge; both responses increased only in fish challenged with the bacterium, regardless of diet. Lysozyme activity increased significantly in fish fed the ED, independent of bacterial exposure. Peroxidase activi ty increased exclusively in fish fed the ED, with no effect from bacterial challenge. These results suggest that the ED is capable of stimulating specific components of the nonspecific immune response in rainbow trout against F. psychrophilum.