Biofilms, microbial communities enclosed in the self-produced extracellular matrix, have a significant impact on human health, environment, and industry. The pathogen Staphylococcus aureus (S. aureus) is recognized as one of the most frequent causes of biofilm-related infections. Phenol-soluble modulins (PSMs) serve as a crucial component, fortifying S. aureus biofilm matrix through self-assembly into amyloid fibrils, which enhances S. aureus colonization and resistance to antibiotics. However, the role of shear rate, one of the critical physiological factors within blood vessels, on the formation of PSM amyloids remains poorly understood. In this work, using a combination of thioflavin T fluorescence kinetic studies, circular dichroism spectrometry, and electron microscopy, we demonstrated that shear rates ranging from 150 to 300 s -1 accelerate fibrillation of PSMα1, α3, and α4 into amyloids, resulting in elongated amyloid structures. Furthermore, PSMα1, α3, and α4 predominantly self-assembled into amyloid fibers with a cross-α structure under shear conditions, deviating from the typical β-sheet configuration of PSM amyloids. These findings imply the role of shear rates within the bloodstream on enhancing PSM self-assembly that is associated with S. aureus biofilm formation.