The transmission of Mycobacterium tuberculosis (TB) requires extensive damage to the lungs to facilitate bacterial release into the airways, and it is therefore likely that the microorganism has evolved mechanisms to exacerbate its local pathology. This study examines the inhibitory effects of lipids extracted and purified chromatographically from TB on the surface-active function of lavaged bovine lung surfactant (LS) and a clinically relevant calf lung surfactant extract (CLSE). Total lipids from TB greatly inhibited the surface activity of LS and CLSE on the pulsating bubble surfactometer at physical conditions applicable for respiration in vivo (37°C, 20cycles/min, 50% area compression). Minimum surface tensions for LS (0.5mg/ml) and CLSE (1mg/ml) were raised from <1mN/m to 15.7±1.2 and 18.7±1.3mN/m after 5min of bubble pulsation in the presence of total TB lipids (0.15mg/ml). TB mixed waxes (0.15mg/ml) and TB trehalose monomycolates (TMMs, 0.15mg/ml) also significantly inhibited the surface activity of LS and CLSE (minimum surface tensions of 10–16mN/m after 5min of bubble pulsation), as did purified trehalose 6,6′-dimycolate (TDM, cord factor). Phosphatidylinositol mannosides (PIMs, 0.15mg/ml) from TB had no inhibitory effect on the surface activity of LS or CLSE. Concentration dependence studies showed that LS was also inhibited significantly by total TB lipids at 0.075mg/ml, with a smaller activity decrease apparent even at 0.00375mg/ml. These findings document that TB contains multiple lipids that can directly impair the biophysical function of endogenous and exogenous lung surfactants. Direct inhibition by TB lipids could worsen surfactant dysfunction caused by plasma proteins or other endogenous substances induced by inflammatory injury in the infected lungs. TB lipids could also inhibit the effectiveness of exogenous surfactants used to treat severe acute respiratory failure in TB patients meeting criteria for clinical acute lung injury (ALI) or the acute respiratory distress syndrome (ARDS).