Nitric oxide produced by three different isoforms of nitric oxide synthase (NOS) widely expressed in virtually all vascular cell types is mostly produced by the endothelial isoform (eNOS) in endothelial cells where it plays a crucial role in vascular tone and structure regulation. It also exerts an anti-inflammatory influence, inhibits platelets adhesion and aggregation, and prevents smooth muscle cells proliferation and migration. Several lines of evidence link endothelial dysfunction, characterized by decreased bioavailability of nitric oxide, with the development of many pathological conditions such as heart failure, hypertension, diabetes and atherosclerosis. This review focuses on nitric oxide-dependent endothelial dysfunction in cardiovascular diseases, its clinical detection and relevance, potential pathogenic mechanisms and possible therapies.