Over the past six decades, the folate biosynthetic pathway has provided a rich source of drug targets for the treatment of proliferative diseases. Drugs targeting dihydrofolate reductase have been especially successful as anticancer (methotrexate), antibacterial (trimethoprim, TMP) and antiprotozoal (cycloguanil, pyrimethamine) therapeutics. While trimethoprim remains a clinically important antimicrobial DHFR inhibitor, resistance by point mutations in otherwise sensitive strains as well as the natural insensitivity of several species limits its use. In this review, an historical overview of the attempts to develop drugs that target the folate pathway is presented along with a discussion of the basis of insensitivity to trimethoprim. From this vantage, we have developed the propargyl-linked antifolates as potent inhibitors of TMP-insensitive enzymes and strains. The structural basis of the increased affinity is detailed to promote the development of further generations of antifolates.