Malnutrition remains a pressing global issue, especially in low-income regions where limited access to affordable animal protein exacerbates food insecurity. Aquaculture offers a sustainable solution by providing a reliable source of animal protein, yet its rapid expansion introduces challenges such as disease outbreaks, antibiotic overuse, and environmental degradation. Enhancing fish health is critical to ensuring the sectors long-term sustainability and contribution to food security. The gut microbiota of fish plays a pivotal role in immunity, nutrient absorption, and disease resistance, with microbial colonization beginning during early larval stages and shaped by diet, habitat, and environmental conditions. This chapter aims to explore the functional role of gut microbiota in fish health and disease prevention; examine dietary strategies such as probiotics, prebiotics, and short-chain fatty acids (SCFAs) that modulate gut microbiota and reduce antibiotic reliance; highlight the application of metagenomic tools for analyzing gut microbial communities; and discuss future directions in microbiome-based approaches for sustainable aquaculture. Advances in next-generation sequencing and integrative omics have revolutionized microbiome research, offering insights into microbial diversity and function. These emerging tools and strategies present transformative potential to promote fish health, mitigate environmental risks, and advance sustainable aquaculture to ensure global food and nutritional security.