| Summary: | Postbiotics are innovative tools in animal husbandry, providing eco-friendly solutions for disease management within the industry. In this study, a new postbiotic product was evaluated for its impact on the health of rainbow trout (<i>Oncorhynchus mykiss</i>). In vivo studies were conducted to assess the safety of the <i>Weissella cibaria</i> strains used in postbiotic production. Additionally, this study evaluated the impact of diet supplementation with 0.50% postbiotics on growth performance during a 30-day feeding trial; the gut microbial communities, immunomodulation, and protection against <i>Yersinia ruckeri</i> infection were evaluated. The strains did not harm the animals during the 20-day observation period. Furthermore, the effect of postbiotics on growth performance was not significant (<i>p</i> < 0.05). The treated group showed a significant increase in acid-lactic bacteria on the 30th day of the feeding trial, with counts of 3.42 ± 0.21 log CFU/mL. Additionally, there was an up-regulation of the pro-inflammatory cytokine <i>IL-1β</i> in head kidney samples after 48 h of feed supplementation, whereas cytokines <i>IL-10</i>, <i>IL-8, INF-γ</i>, and <i>TNF-α</i> were down-regulated. The findings indicate that rainbow trout fed with postbiotics saw an improvement in their survival rate against <i>Y. ruckeri</i>, with a 20.66% survival improvement in the treated group. This study proves that incorporating postbiotics from two strains of <i>W. cibaria</i> previously isolated from rainbow trout into the diet of fish has immunomodulatory effects, enhances intestinal microbial composition, and improves fish resistance against <i>Y. ruckeri.</i>
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