Biofilms alter their metabolism in response to environmental stress. This study explores the effect of a hyperosmotic agent-antibiotic treatment on the metabolism of Staphylococcus aureus biofilms through the use of nuclear magnetic resonance (NMR) techniques. To determine the metabolic activity of S. aureus, we quantified the concentrations of metabolites in spent medium using high-resolution NMR spectroscopy. Biofilm porosity, thickness, biovolume, and relative diffusion coefficient depth profiles were obtained using NMR microimaging. Dissolved oxygen concentration was measured to determine the availability of oxygen within the biofilm. Under vancomycin-only treatment, the biofilm communities switched to fermentation under anaerobic condition, as evidenced by high concentrations of formate (7.4 +/- 2.7mM), acetate (13.1 +/- 0.9mM), and lactate (3.0 +/- 0.8mM), and there was no detectable dissolved oxygen in the biofilm. In addition, we observed the highest consumption of pyruvate (0.19mM remaining from an initial 40mM concentration), the sole carbon source, under the vancomycin-only treatment. On the other hand, relative effective diffusion coefficients increased from 0.73 +/- 0.08 to 0.88 +/- 0.08 under vancomycin-only treatment but decreased from 0.71 +/- 0.04 to 0.60 +/- 0.07 under maltodextrin-only and from 0.73 +/- 0.06 to 0.56 +/- 0.08 under combined treatments. There was an increase in biovolume, from 2.5 +/- 1mm(3) to 7 +/- 1mm(3), under the vancomycin-only treatment, while the maltodextrin-only and combined treatments showed no significant change in biovolume over time. This indicated that physical biofilm growth was halted during maltodextrin-only and combined treatments.