Dihydrofolate reductase (DHFR) plays an essential role in cellular biochemistry and has been a well-recognized drug target for over fifty years. Antifolate inhibitors of DHFR, including clinically used therapeutics such as methotrexate, trimethoprim, and pyrimethamine have been successful as anticancer, antibacterial, antifungal and antiparasitic agents. As resistant strains of these microorganisms evolve and as new disease threats arise, the need for new antifolates that are potent and specific for infectious organisms becomes more pressing. Several new antifolates have been reported over the past decade; many of these are potent against a particular species of DHFR, but achieving the goal of potency and selectivity has proven to be more difficult. This review will describe recent advances in attaining species selectivity in developing new antifolates. Specifically, advances in developing inhibitors against Pneumocystis jirovecii and Plasmodium falciparum, the causative agents in pneumocystis pneumonia and malaria, respectively, will be presented.