In prokaryotes and eukaryotes, folate (vitamin B9) is an essential metabolic cofactor required for all actively growing cells. Specifically, folate serves as a one-carbon carrier in the synthesis of amino acids (such as methionine, serine, and glycine), N-formylmethionyl-tRNA, coenzyme A, purines and thymidine. Many microbes are unable to acquire folates from their environment and rely on de novo folate biosynthesis. In contrast, mammals lack the de novo folate biosynthesis pathway and must obtain folate from commensal microbiota or the environment using proton-coupled folate transporters. The essentiality and dichotomy between mammalian and bacterial folate biosynthesis and utilization pathways make it an ideal drug target for the development of antimicrobial agents and cancer chemotherapeutics. In this minireview, we discuss general aspects of folate biosynthesis and the underlying mechanisms that govern susceptibility and resistance of organisms to antifolate drugs.