Aquaculture sustainability requires a reduction in the reliance on marine-derived raw materials such as fish oil in aquafeeds while maintaining fish health and product quality. This study investigated the effects of replacing fish oil with plant oils supplemented with DHA-rich Schizochytrium limacinum biomass on the gut microbiota of European sea bass (Dicentrarchus labrax). S. limacinum SR21—an oleaginous microalga naturally rich in omega-3 fatty acids—was produced through heterotrophic fermentation using crude glycerol, a waste stream from biodiesel production, within a circular economy framework. A 21-week feeding trial was conducted in an indoor recirculating aquaculture system using 280 fish distributed across eight tanks. Four experimental diets were tested: fish oil-based (FO), plant oil-based without microalga (VO + 0), and plant oil-based supplemented with 5% (VO + 5) or 10% (VO + 10) microalgal biomass. Gut microbiota was analyzed in 22 fish per group using 16S rRNA gene sequencing. While alpha and beta diversity analyses of gut microbiota revealed modest structural shifts at phylum and class ranks, genus-rank differences were evident, with Lactobacillus and Clostridium sensu stricto associated with FO and VO + 0 diets, and Pseudomonas and Staphylococcus enriched in microalga-supplemented groups. Functional inference highlighted enhanced bile acid biosynthesis and carbohydrate metabolism in VO + 0, whereas antioxidant-related pathways, including ubiquinone and carotenoid biosynthesis, were stimulated in VO + 5 and VO + 10 groups. These results demonstrate that S. limacinum biomass modulates microbiota functional capacity, potentially contributing to oxidative stress mitigation and host resilience. The findings support microbiota-informed feed formulation strategies to advance sustainable aquaculture.