Although nonsteroidal anti-inflammatory drugs (NSAIDs) target primarily cyclooxygenase enzymes, a subset of NSAIDs containing carboxylate groups also has been reported to competitively inhibit dihydrofolate reductase (DHFR). In this study, we have characterized NSAID interactions with human DHFR based on kinetic, NMR, and X-ray crystallographic methods. The NSAIDs target a region of the folate binding site that interacts with the p-aminobenzoyl-l-glutamate (pABG) moiety of folate and inhibit cooperatively with ligands that target the adjacent pteridine-recognition subsite. NSAIDs containing benzoate or salicylate groups were identified as having the highest potency. Among those tested, diflunisal, a salicylate derivative not previously identified to have anti-folate activity, was found to have a Ki of 34 μM, well below peak plasma diflunisal levels reached at typical dosage levels. The potential of these drugs to interfere with the inflammatory process by multiple pathways introduces the possibility of further optimization to design dual-targeted analogs.