Misuse and overuse of antibiotics in aquaculture has proven to be an unsustainable practice leading to increased bacterial resistance. An alternative strategy involves the inclusion of immunostimulants in fish diets, especially fungal and herbal compounds already authorized for human consumption, hence without environmental or public health concerns. In this study, we used a holistic and cross-disciplinary pipeline to assess the immunostimulatory properties of two fungi: Trametes versicolor and Ganoderma lucidum; one herbal supplement, capsaicin in the form of Espelette pepper (Capsicum annuum), and a combination of these fungal and herbal additives on rainbow trout (Oncorhynchus mykiss). We investigated the impact of diet supplementation for 7 weeks on survival, growth performance, cellular, humoral, and molecular immune parameters, as well as the intestinal microbial composition of the fish. Uptake of herbal and fungal compounds influenced the expression of immune related genes, without generating an inflammatory response. Significant differences were detected in the spleen-tlr2 gene expression. Supplementation with herbal additives correlated with structural changes in the fish intestinal microbiota and enhanced overall intestinal microbial diversity. Results demonstrated that the different treatments had no adverse effect on growth performance and survival, suggesting the safety of the different feed additives at the tested concentrations. While the mechanisms and multifactorial interactions remain unclear, this study provides insights not only in regard to nutrition and safety of these compounds, but also how a combined immune and gut microbiota approach can shed light on efficacy of immunostimulant compounds for potential commercial inclusion as feed supplements.