Understanding the environmental factors that affect the production of virulence factors has major implications in evolution and medicine. While spatial structure is important in virulence factor production, observations of this relationship have occurred in undisturbed or continuously disturbed environments. However, natural environments are subject to periodic fluctuations, including changes in physical forces, which could alter the spatial structure of bacterial populations and impact virulence factor production. Using Pseudomonas aeruginosa PA14, we periodically applied a physical force to biofilms and examined production of pyoverdine. Intermediate frequencies of disturbance reduced the amount of pyoverdine produced compared to undisturbed or frequently disturbed conditions. To explore the generality of this finding, we examined how an intermediate disturbance frequency affected pyoverdine production in 21 different strains of P. aeruginosa. Periodic disturbance increased, decreased, or did not change the amount of pyoverdine produced relative to undisturbed populations. Mathematical modeling predicts that interactions between pyoverdine synthesis rate and biofilm density determine the amount of pyoverdine synthesized. When the pyoverdine synthesis rates are high, depletion of the biofilm due to disturbance reduces the accumulation of pyoverdine. At intermediate synthesis rates, production of pyoverdine increases during disturbance as bacteria dispersed into the planktonic state enjoy increased growth and pyoverdine production rates. At low synthesis rates, disturbance does not alter the amount of pyoverdine produced since disturbance-driven access to nutrients does not augment pyoverdine synthesis. Our results suggest that environmental conditions shape robustness in the production of virulence factors and may lead to novel approaches to treat infections.