Chilean salmon production is significantly affected by infectious fish diseases, which negatively impact growth and performance and are associated with increased mortality. Among these, piscirickettsiosis, caused by Piscirickettsia salmonis, is the most significant, accounting for approximately 44.7% and 21.7% of total infectious mortality in Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) production in 2023. The limited efficacy of current vaccines against this disease has led to an overreliance on antibiotics, raising environmental, health, and commercial concerns. This scenario has generated a growing need to seek alternative non-pharmacological strategies to reduce antibiotic use. To achieve this, it is necessary to develop environmentally friendly products and formulations that protect the sustainability of the salmon industry. In this sense, probiotics can be applied to salmon to improve fish health, growth, and disease resistance, and potentially enhance the environmental sustainability of farming practices. The objective of this study was to evaluate a functional probiotic formulation enriched with antibacterial peptides (BioProtein, Chile), designed to modulate the immune response in fish and act directly against P. salmonis. Preliminary, in vitro results demonstrated that 6.5 mg/mL of antimicrobial peptides inhibited the growth of P. salmonis cultured in broth medium, suggesting a direct action of the product on this bacterium. Furthermore, the antimicrobial effect of the probiotic formulation was determined using the SHK-1 cell line, derived from head kidney of S. salar, which exhibits monocyte-macrophage characteristics and represents the cellular target of P. salmonis in salmonids. The results obtained showed an inhibition of the P. salmonis infection process, evaluated by the cytotoxicity induced by bacterial infection in SHK1 cells. This effect was obtained using P. salmonis genogroup LF89 and EM90 during the infection, using a probiotic dose range of 1–500 µg/mL. These findings suggest the potential application of this probiotic formulation enriched with antimicrobial peptides as a functional additive against P. salmonis in fish diets.