Used primarily to treat and prevent protozoal infections, particularly malaria and toxoplasmosis. It works by inhibiting dihydrofolate reductase, an enzyme essential for parasite DNA synthesis. Often combined with sulfadoxine or other sulfonamides for malaria treatment to enhance efficacy and delay resistance.

In immunocompromised patients, such as those with HIV/AIDS, it is used in combination with sulfadiazine and folinic acid to manage active toxoplasmosis. Also used in veterinary medicine for prevention and treatment of coccidiosis in animals.

Due to its antifolate activity, it requires careful monitoring to avoid bone marrow suppression and other hematologic side effects.