Bacillus subtilis is a spore-forming microorganism, recognized as a safe probiotic strain. Its endospores are easily produced at a large scale, can be dehydrated and maintain their characteristics after long-term storage, providing great advantages for their application in aquafeeds. Nonetheless, knowledge on the effects that B. subtilis provokes on fish mucosal immunity is still scarce. In this context, the aim of this work was to explore the effects of the oral administration of B. subtilis spores to rainbow trout (Oncorhynchus mykiss) for 30 days on the intestinal and skin mucus, focusing on a range of immune and enzymatic parameters. Thus, fish supplemented with B. subtilis spores showed increased levels of total immunoglobulin (Ig) in intestinal and skin mucus, as well as IgM levels in both mucus and serum. B. subtilis spores induced an increase of peroxidase activities in mucus from both sources. Although superoxide dismutase (SOD) activity was not affected by the spores, NO production significantly decreased in skin mucus. Finally, the bactericidal activity of the intestinal and skin mucus was significantly higher in fish fed the probiotic spores. The results obtained demonstrate that the dietary supplementation with B. subtilis spores enhances mucosal defense mechanisms by increasing immunological parameters of the intestinal and skin mucus.