Pulmonary surfactant (PS) is a lipid-protein complex essential to stabilise the delicate structure of mammalian alveoli along with successive compression-expansion respiratory cycles. To do so, surfactant reduces dramatically surface tension at the air-liquid interface, an activity that depends critically on a proper lipid composition and the presence of some specific surfactant proteins. Lack or dysfunction of this system is associated with severe respiratory pathologies, which are in some cases treated by supplementation with exogenous surfactant materials. The biophysical function and performance of PS, in health and disease, are directly influenced by its composition, structure and mechanical properties. This review summarises the main biophysics concepts behind the mechanisms that define surfactant function in a healthy lung and in pathological situations. It also revises some of the most useful biophysical techniques that provide information about surfactant-related processes. Finally, translational biophysics will be invoked to illustrate how biophysical studies may contribute to understand the role of surfactant in health and disease and to design better surfactant-based therapeutic approaches.