Benzothiazole derivatives are an important class of heterocyclic compounds with well-documented anticancer properties. Herein, we report the synthesis, characterization, and anticancer screening of a novel series of 2,6-disubstituted benzothiazole derivatives. The target compounds were synthesized through condensation and oxidative cyclization of substituted benzaldehydes with 2-aminothiophenol, followed by electrophilic substitution at the 6-position. The synthesized compounds were characterized using NMR, IR, and HRMS techniques. Their anticancer potential was evaluated against MCF-7 (breast), HeLa (cervical), and A549 (lung) human cancer cell lines using the MTT assay. Among the series, compound 1d exhibited the highest cytotoxic activity with IC₅₀ values in the low micromolar range. These findings indicate that 2,6-disubstituted benzothiazoles are promising candidates for further development as anticancer agents.