Dihydrofolate reductase (DHFR) is an enzyme involved in the cellular synthesis of DNA building blocks, such as thymidylate, and is critical for cell proliferation. Therefore, DHFR was recognized as a valid anticancer target; its classical inhibitors, aminopterin and methotrexate (MTX), demonstrated clinical effectiveness in anticancer therapy and opened the cancer chemotherapy era. Despite significant achievements in chemotherapeutic applications of DHFR inhibitors and the development of new effective drugs, such as pralatrexate and trimetrexate, cancer cell resistance emerged, encouraging the search for new effective drugs with alternative cellular transport and metabolism. The prospective research directions focus on applications of different scaffolds in drug design, developing multitargeted antifolates, and exploiting radiosensitizing properties.