Staphylococcus aureus uses quorum sensing and nutrient availability to control the expression of -regulated virulence factors. Quorum sensing is mediated by autoinducing peptide (AIP), which at a high concentration reduces expression of surface attachment proteins (, ) and increases expression of exotoxins () and proteases (). Nutrient availability can be sensed through the / system. Low nutrients increase expression of , which augments expression of and , distinct from the activity of AIP. The formation of spatial structure, such as biofilms, can alter quorum sensing and nutrient acquisition. In natural environments, biofilms encounter forces that may alter their spatial structure. These forces may impact quorum sensing and/or nutrient acquisition and thus affect the expression of -regulated virulence factors. However, this has not been studied. We show that periodically disturbing biofilms composed of S. aureus using a physical force affected the expression of -regulated virulence factors. In nutrient-poor environments, disturbance increased the expression of , , , and Disturbance in a nutrient-rich environment at low or high disturbance amplitudes moderately reduced expression of and but increased expression of and . Interestingly, at an intermediate amplitude, the overall expression of -regulated virulence factors was the lowest; expression of and remained unchanged relative to an undisturbed biofilm, while expression of and significantly decreased. We hypothesize that these changes are a result of disturbance-driven changes in access to AIP and nutrients. Our results may allow the identification of environments where virulence is enhanced, or reduced, owing to a disturbance. Bacteria, such as Staphylococcus aureus, integrate signals from the environment to regulate genes encoding virulence factors. These signals include those produced by quorum-sensing systems and nutrient availability. We show that disturbing the spatial organization of S. aureus populations can lead to changes in the expression of virulence factors, likely by altering the ways in which S. aureus detects these signals. Our work may allow us to identify environments that increase or reduce the expression of virulence factors in S. aureus.