Hyperosmotic agents such as maltodextrin negatively impact bacterial growth through osmotic stress without contributing to drug resistance. We hypothesized that a combination of maltodextrin (osmotic agent) and vancomycin (antibiotic) would be more effective against Staphylococcus aureus biofilms than either alone. To test our hypothesis, S. aureus was grown in a flat plate flow cell reactor. Confocal laser scanning microscopy images were analyzed to quantify changes in biofilm structure. We used dissolved oxygen microelectrodes to quantify how vancomycin and maltodextrin affected the respiration rate and oxygen penetration into the biofilm. We found that treatment with vancomycin or maltodextrin altered biofilm structure. The effect on the structure was significant when they were used simultaneously to treat S. aureus biofilms. In addition, vancomycin treatment increased the oxygen respiration rate, while maltodextrin treatment caused an increase and then a decrease. An increased maltodextrin concentration decreased the diffusivity of the antibiotic. Overall, we conclude that (1) an increased maltodextrin concentration decreases vancomycin diffusion but increases the osmotic effect, leading to the optimum treatment condition, and (2) the combination of vancomycin and maltodextrin is more effective against S. aureus biofilms than either alone. Vancomycin and maltodextrin act together to increase the effectiveness of treatment against S. aureus biofilm growth. Biotechnol. Bioeng. 2015;112: 2562-2570. (c) 2015 Wiley Periodicals, Inc.