Nitric oxide synthases (NOS) are crucial enzymes responsible for the production of nitric oxide (NO), a signaling molecule with essential roles in vascular regulation, immune defense, and neurotransmission. The three NOS isoforms, endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS), are tightly regulated by inflammatory mediators and cellular signaling pathways. While physiological NO production is vital for maintaining homeostasis, dysregulated NOS activity contributes to the pathogenesis of numerous diseases, including cardiovascular disorders, neurodegenerative conditions, and cancer. Recent advances in understanding the molecular mechanisms of NOS regulation have unveiled novel therapeutic opportunities, including isoform-specific modulators, upstream pathways, and nanotechnology-enhanced delivery systems. This review highlights these advancements, offering insights into how targeting NOS and its regulatory network can enable precise and effective therapeutic strategies for managing inflammation-driven pathologies.