Dihydrofolate reductase (DHFR), a ubiquitous oxidoreductase, occupies a pivotal position in folate metabolism. This review presents a comprehensive analysis of DHFR's structure, catalytic mechanism, and diverse physiological roles. We highlight its essential function in purine and thymidylate biosynthesis, emphasizing its impact on DNA replication and cell proliferation. The review further explores the multifaceted implications of DHFR in disease: its exploitation in cancer chemotherapy through targeted inhibition, its role in bacterial antibiotic resistance, and potential associations with autoimmune disorders. Finally, we discuss emerging research avenues delving into novel DHFR inhibitors for cancer therapy, strategies to combat DHFR-mediated antibiotic resistance, and the exploration of DHFR gene polymorphisms in personalized medicine approaches for autoimmune diseases. This review underscores the critical role of DHFR in both normal cellular processes and various disease states, paving the way for future advancements in targeted therapeutic interventions.