<p>Aquaculture is a cornerstone in protein production with the cultivation of Nile tilapia (<i>Oreochromis niloticus</i>), but it has faced challenges from pathogens such as the emerging bacterium <i>Lactococcus garvieae.</i> Humic acid (HA) was used as a prophylactic measure to evaluate its effect on the intestinal microbiota, immune system, and survival in tilapia challenged with <i>L. garvieae</i> under laboratory conditions. Four concentrations of HA were tested in feed over 83&#xa0;days. The intestinal microbiota was analyzed in treatment IV (1.0&#xa0;g/kg of feed) and control through 16S rRNA gene sequencing (V3). Liver and gill tissue were collected for immune system gene expression analysis (COX-2, TGF, IL-1B, and TNF-α) through real-time qPCR methods. On day 71, the fish were inoculated by immersion with <i>L. garvieae</i> (CL₅₀ = 8.5 × 10⁵&#xa0;CFU/mL). Gene expression in the liver (COX-2 and TNF-α) and gills (COX-2, TGF-β, and IL-1β) increased significantly in treatment IV. <i>Cetobacterium</i> was among the most abundant genera in the tilapia gut. Fusobacteriota (phylum) and the genera <i>Aeromonas</i>, <i>Rhodobacter</i>, <i>Gemobacter</i>, and <i>Ideonella</i> were enriched in HA, whereas the phylum Patescibacteria and genera <i>Pelomonas</i>, <i>Nitrobacter</i>, and <i>Clostridioides</i> decreased significantly. Dietary HA supplementation significantly altered gut microbiota diversity and the interaction network core. Humic acid supplementation significantly improved tilapia survival against <i>L. garvieae</i> by enhancing immune gene expression and modulating gut microbiota—enriching Fusobacteriota and beneficial genera (<i>Rhodobacter</i>, <i>Ideonella</i>) while reducing pathogens (<i>Pelomonas</i>, <i>Clostridioides</i>). These findings support HA as a prophylactic alternative against lactococcosis in Nile tilapia (<i>O. niloticus</i>) farming.</p>