Nitric oxide (NO), the highly reactive radical gas, provides an attractive strategy in the control of microbial infections. NO not only exhibits bactericidal effect at high concentrations but also prevents bacterial attachment and disperses biofilms at low, nontoxic concentrations, rendering bacteria less tolerant to antibiotic treatment. The endogenously generated NO by airway epithelium in healthy populations significantly contributes to the eradication of invading pathogens. However, this pathway is often compromised in patients suffering from chronic lung infections where biofilms dominate. Thus, exogenous supplementation of NO is suggested to improve the therapeutic outcomes of these infectious diseases. Compared to previous reviews focusing on the mechanism of NO-mediated biofilm inhibition, this review explores the applications of NO for inhibiting biofilms in chronic lung infections. It discusses how abnormal levels of NO in the airways contribute to chronic infections in cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and primary ciliary dyskinesia (PCD) patients and why exogenous NO can be a promising antibiofilm strategy in clinical settings, as well as current and potential in vivo NO delivery methods.