Materials formed with a base of silk fibroin (SF) are successfully used in tissue engineering since their properties are similar to those of natural extracellular matrixes. Mixing SF with different polymers, for example, polyethylene oxide (PEO) and polyvinylpyrrolidone (PVP), allows the production of fibers, hydrogels, and films and their morphology to be controlled. The impact of PEO and PVP on formation and structure of SF adsorption layers was studied at different was studied at different polymer concentrations (from 0.002 to 0.5 mg/mL) and surface lifetimes. The protein concentration was fixed at 0.02 and 0.2 mg/mL. These concentrations are characterized by different types of spontaneously formed structures at the air-water interface. Since both synthetic polymers possess surface activity, they can penetrate the fibroin adsorption layer, leading to a decrease in the dynamic surface elasticity at almost constant surface tension and a decrease in ellipsometric angle Δ and adsorption layer thickness. As shown by AFM, the presence of polymers increases the porosity of the adsorption layer, due to the possible arrangement of protein and polymer molecules into separate domains, and can result in various morphology types such as fibers or tree-like ribbons. Therefore, polymers like PEO and PVP can be used to regulate the SF self-assembly at the interface, which in turn can affect the properties of the materials with high surface areas like electrospun matts and scaffolds.