One of the initial barriers encountered by the airborne nanoparticles (NPs) upon entry into the body is the lung surfactant (LS), which plays a vital role in maintaining the normal respiratory cycle of the lungs. Interaction between gold nanoparticles (AuNPs), which are commonly used in many areas, especially in pharmaceutical applications due to their functional features and LS has been evaluated by combining Langmuir Trough (2D) and Pendant-Drop tensiometer (3D) approaches. AuNPs were synthesized via Turkevich method and AuNP clusters were prepared by using dithiothreitol (DTT) with the different concentrations. Model LS was extracted from a bovine lung and brought into vesicular form after purification. Our results showed that AuNP-DTT clusters are able to penetrate into the LS monolayer and vesicles, change the normal behavior of air-buffer interface, organization of the molecules and phase behavior of the system. In 2D studies, it is seen that the interaction of AuNPs and AuNP-DTT clusters decreases the surface pressure of phase transition region which indicates decreasing the elasticity with a similar manner for first 3 h. In the longer periods, the monolayer presented an increased resistance to compression, indicating the low compressibility of monolayer. After 7 h interaction with AuNP-DTT clusters, at the same surface area (48 cm2), the π of LS films can just reach ∼30 mN·m−1 instead of ∼45 mN·m−1 which proves the induction of the isotherm to form a more disordered structure with lower cohesion between the molecules. In 3D studies, it is found that there becomes a competitive adsorption between LS vesicles and AuNP-DTT clusters in the long-term interaction which may enhance the pulmonary toxicity of the inhaled nanostructure. The ultimate insight provided by this study is to evaluate the toxicity of the interaction of asymmetrical NPs on biological barriers with a realistic approach. As a result of our investigations, it is proved that long-term exposure of anisotropic clusters can cause potential adverse effects on the healthy environment and breathing cycle of lungs more than smaller individual counterparts with the same concentration.