Metal-based nanomaterials (MNMs) represent a large category of the engineered nanomaterials, and have been extensively used to enhance the electrical, optical, and magnetic properties of nanoenabled consumer products. Inhaled MNMs can penetrate deeply into the peripheral lung at which they first interact with the pulmonary surfactant (PS) lining of alveoli. Here we studied the biophysical inhibitory potential of representative MNMs on a modified natural PS, Infasurf, using a novel in vitro experimental methodology called the constrained drop surfactometry (CDS). It was found that the biophysical inhibitory potential of six MNMs on Infasurf ranks in the order CeO2 > ZnO > TiO2 > Ag > Fe3O4 > ZrO2-CeO2. This rank of in vitro biophysical inhibition is in general agreement with the in vitro and in vivo toxicity of these MNMs. Directly imaging the lateral structure and molecular conformation of the PS film using atomic force microscopy revealed that there exists a correlation between biophysical inhibition of the PS film by the MNMs and their aggregation state at the PS film. Taken together, our study suggests that the nano-bio interactions at the PS film are determined by multiple physicochemical properties of the MNMs, including not only well-studied properties such as their chemical composition and particle size, but also properties such as hydrophobicity, dissolution rate, and aggregation state at the PS film found here. Our study provides novel insight into the understanding of nanotoxicology and metallomics of MNMs.