Type II topoisomerases are essential enzymes that modulate the topological state of DNA supercoiling in all living organisms. These enzymes alter DNA topology by performing double-stranded passage reactions on over- or underwound DNA substrates. This strand passage reaction generates a transient covalent enzyme–cleaved DNA structure known as the cleavage complex. Al-though the cleavage complex is a requisite catalytic intermediate, it is also intrinsically dangerous to genomic stability in biological systems. The potential threat of type II topoisomerase function can also vary based on the nature of the supercoiled DNA substrate. During essential processes such as DNA replication and transcription, cleavage complex formation can be inherently more dangerous on overwound versus underwound DNA substrates. As such, it is important to understand the profound effects that DNA topology can have on the cellular functions of type II topoisomerases. This review will provide a broad assessment of how human and bacterial type II topoisomerases recognize and act on their substrates of various topological states.