The macromolecule that carries genetic information, DNA, is considered as an exceptional target for diseases depending on cellular division of malignant cells (<i>i.e.</i> cancer), microbes (<i>i.e.</i> bacteria) or parasites (<i>i.e.</i> protozoa). To aim for a comprehensive review to cover all aspects related to DNA targeting would be an impossible task and, hence, the objective of the present review is to present, from a medicinal chemistry point of view, recent developments of compounds targeting the minor groove of DNA. Accordingly, we discuss the medicinal chemistry aspects of heterocyclic small-molecules binding the DNA minor groove, as novel anticancer, antibacterial and antiparasitic agents.