Dihydrofolalate reductase (DHFR) inhibitors are of significant interest as potential tool against parasites and other microbial infections. Dihydrofolate reductase is a ubiquitous enzyme present in almost all eukaryotic and prokaryotic cells and has potential importance in biochemistry and medicinal chemistry. It catalyses the various biochemical reactions are of vital importance, inhibition of dihydrofolatereductase plays important role in controlling growth of various parasites. Inhibition of dihydrofolate reductase becomes very useful tool for controlling number of diseases and disorders like cancer, malaria, toxoplasmosis, rheumatoid arthritis and tuberculosis. The various drugs are also available which are based on inhibition of dihydrofolate reductase. This article presents overview on various potential applications of dihydrofolate reductase inhibitors.