The quest for new anticancer agents remains one of the foremost challenges in medicinal chemistry and pharmaceutical sciences. Heterocyclic compounds, particularly benzothiazole derivatives, have emerged as a versatile class of molecules exhibiting significant pharmacological activities. Among them, 2,6-disubstituted benzothiazoles have received considerable attention due to their promising cytotoxic effects against a broad range of human cancer cell lines. Substituents at the C-2 and C-6 positions critically influence the electronic distribution, molecular recognition, and interactions with biological targets such as DNA, topoisomerases, kinases, and mitochondrial enzymes.

This review comprehensively discusses recent advances in the synthesis, structural modifications, and anticancer evaluations of 2,6-disubstituted benzothiazole derivatives. Special emphasis is placed on synthetic methodologies ranging from classical condensation reactions to modern cross-coupling and green chemistry approaches. The review also outlines the structure–activity relationship (SAR), mechanistic pathways underlying cytotoxicity, and the role of substituents in enhancing anticancer activity. Furthermore, we highlight challenges in clinical translation, pharmacokinetics, and future perspectives involving nanotechnology-based formulations and computational modeling.