Functional feeds, as a prophylactic strategy, are a promising alternative to address stressful production-related activities (e.g., seawater transfer, delousing) and infectious diseases in salmon farming. To understand the effect of Paecilomyces variotii on short-term growth performance and health responses of vaccinated Atlantic salmon pre-smolts, a control diet (D1) and three experimental diets, where P. variotii replaced 5% (D2), 10% (D3) or 20% (D4) of crude protein, were fed to fish for 28 days in freshwater. Fish fed diets containing P. variotii had a significant dose-dependent linear improvement in feed conversion ratio. Also, D4 group showed a gene expression related to signal truncation and gut homeostasis, while in head kidney (HK), P. variotii activated and controlled immune responses through c-type lectin receptor, downstream signalling molecules (myd88, tollip), cytokines (tnfα, il1β, ifnγ), and effector molecules (cath-2, c3, prx). Moreover, an upregulation of antigen presenting cell markers (mhcii, cd83) and T cell transcriptional factors (gata3, rorc, foxp3) was detected in HK, suggesting that P. variotii could coordinate the innate and adaptive mechanisms. Interestingly, D2 increased specific IgM against Vibrio anguillarum in vaccinated salmon. KEGG analysis revealed that D4 induced decreased abundance of proteins related to inflammatory pathways, e.g., like salmonella infection, apoptosis and necroptosis as well as innate and adaptive signalling pathways in the HK. On the contrary, D4 induced high abundance of proteins related to these inflammatory pathways in the skin mucus (Skm). In addition, complement proteins (i.e., C1q, C4, C7) and arginine metabolism were also in high abundance in the SKm. In relation to the gut microbiota, fish fed D2 and D3 showed low abundance of key lactic acid bacteria (e.g., Weisella, Leuconostoc, Lactobacillus) but high abundance Photobacterium and Ligilactobacillus compared with D1 in the gut. Overall, feed inclusion of P. variotii improved fish growth performance and modulated health response in Atlantic salmon pre-smolts.