Osmolyte interactions with ligands can affect their affinity for proteins and are dependent upon the cosolute and the functional groups of the ligand. Here, we explored ligand binding to Bacillus anthracis dihydropteroate synthase (BaDHPS) under osmotic stress conditions. Osmolyte effects were specific to the cosolute and ligand, suggesting interaction of the osmolytes with the free ligands in solution. The association rates of pterin pyrophosphate were mostly unaffected by the osmolytes, except for a 2-fold decrease in the presence of 1 M trehalose, while the dissociation rates decreased in most osmolyte solutions. The viscosity and dielectric constant of the solution did not correlate with the effects of the osmolytes. Experimental results were compared with predicted preferential interaction coefficients (Delta mu(23)/RT) between the osmolytes and ligands. The Delta mu(23)/RT were able to predict the experimental data for most of the osmolytes. Trehalose and proline effects did not correlate with the predicted values, indicating that these two osmolytes may affect binding in more complex ways than simple preferential interactions. Additionally, osmolytes weakly interacted with the sulfa drug sulfathiazole, which altered its affinity for BaDHPS, suggesting that these types of weak interactions can also impact drug binding. As osmolytes affect ligands binding to two different folate cycle enzymes (DHFRs and DHPS), we predicted how ligand binding to other folate cycle enzymes will be altered by the presence of osmolytes.