Fish are constantly exposed to various bacterial, fungal, and viral pathogens in both natural and artificial environments. As the first line of defense against these external threats, mucosal surfaces, particularly skin mucus, play a critical role. Skin mucus is not only a physical barrier but also an active immunological interface enriched with molecules such as lysozyme, immunoglobulins, complement proteins, and antimicrobial peptides. In aquaculture systems, infectious diseases represent a major threat to fish health and productivity. This study aimed to evaluate the immunological components and antimicrobial activity of skin mucus obtained from Oncorhynchus mykiss (O. mykiss). Levels of immunoglobulin M (IgM), complement components C3 and C4 were quantified using enzyme-linked immunosorbent assay (ELISA). Lysozyme activity was assessed spectrophotometrically. The antimicrobial properties of the mucus were tested against eight selected bacterial and fungal pathogens by determining their Minimum Inhibitory Concentration (MIC) and Minimum Lethal Concentration (MLC). Skin mucus samples contained significant levels of immune-related proteins. Mucus showed significant antimicrobial activity against Gram-positive and Gram-negative bacteria and some common fungal species. In particular, its bacteriostatic and bactericidal effects are quite high against Gram-negative bacteria. This study demonstrates that the skin mucus of O. mykiss is not only a passive barrier but also an active component of innate immunity, offering potential as a natural biological source in sustainable aquaculture practices and in the development of alternative antimicrobial agents.