<p>Topoisomerases are crucial enzymes that control the DNA topology in replication, transcription, recombination, and chromosomal segregation, and therefore are validated as the targets of anticancer therapy. Topoisomerase-targeting agents are mechanistically divided into poison-type and catalytic-type inhibitors. Poison-type inhibitors stabilize covalent enzyme–DNA cleavage complexes, whereas catalytic inhibitors prevent enzymatic turnover without stabilizing cleavage complexes. Both classes may selectively target either topoisomerase I or topoisomerase II isoforms. Established and clinically useful agents based on camptothecin derivatives, etoposide, anthracyclines, and other related compounds continue to play a central role in chemotherapeutic regimens in solid tumor and hematologic malignancies, albeit with dose limiting toxicity, genotoxic liability, cardiotoxicity (partially caused by Topo IIβ engagement), and drug resistance. Recent medicinal chemistry efforts have reported several next-generation topoisomerase-targeting scaffolds with improved in vitro potency and diverse mechanistic profiles. Molecules based on quinoline, acridine-thiosemicarbazone, pyrazine, pyrimidine, benzimidazole-triazole, indenoisoquinoline, and fluoroquinolone have been reported as effective topoisomerase I and II inhibitors, several of which have an inhibitory activity of nanomolar to low-micromolar. In silico and experimental profiling indicate that the derivatives selected have favourable drug-like characteristics, less susceptible to P-glycoprotein-mediated efflux as well as possibly improved safety-related properties compared to classical topoisomerase poisons. This review summarizes clinically established topoisomerase inhibitors and emerging scaffold-based inhibitors, highlighting structure–activity relationships, mechanistic differences between poison and catalytic inhibitors, resistance mechanisms, and translational challenges in the development of safer and more selective topoisomerase-targeted anticancer agents.</p>