Nitric oxide (NO) is a multifunctional gaseous signaling molecule that plays a central role in maintaining physiological homeostasis while also contributing to diverse pathological processes. Its biological effects are highly context dependent and are shaped by the interplay between nitric oxide synthase (NOS) isoforms, local redox balance, temporal dynamics, cellular environment, and concentration gradients. Under physiological conditions, NO support's vascular function, neuronal communication, and immune regulation through well-coordinated signaling pathways, including cyclic GMP production and post-translational protein modifications. However, under conditions of oxidative stress or sustained inflammatory activation, NO signaling can shift toward the generation of reactive nitrogen species, leading to cellular dysfunction, oxidative injury, and tissue damage. This review highlights the non-redundant roles of NOS isoforms—endothelial (eNOS), neuronal (nNOS), and inducible (iNOS)—as context-specific regulators of NO production, each contributing distinctly to physiological and pathological outcomes. eNOS primarily maintains vascular integrity, nNOS regulates neural signaling, and iNOS drives inflammatory responses, with their dysregulation playing a key role in cardiovascular, neuroinflammatory, oncologic, and renal diseases. Across these systems, the balance between protective and deleterious NO signaling is determined by isoform activity, redox environment, exposure time, and local concentration. Finally, we discuss emerging therapeutic strategies that aim to modulate NO signaling in a more precise and context-aware manner, including isoform-selective targeting, redox modulation, controlled NO delivery systems, and computationally guided drug design. Together, these approaches underscore the importance of integrating mechanistic insight with translational applications to better harness the therapeutic potential of NO while minimizing its cytotoxic effects.