A series of chalcone-amidobenzothiazole conjugates (9a–k and 10a,b) have been synthesized and evaluated for their anticancer activity. All these compounds exhibited potent activity and the IC50 of two potential compounds (9a and 9f) against different cancer cell lines are in the range of 0.85–3.3 μM. Flow cytometric analysis revealed that these compounds induced cell cycle arrest at G2/M phase in A549 cell line leading to caspase-3 dependent apoptotic cell death. The tubulin polymerization assay (IC50 of 9a is 3.5 μM and 9f is 5.2 μM) and immuofluorescence analysis showed that these compounds effectively inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Further, Annexin staining also suggested that these compounds induced cell death by apoptosis. Moreover, docking experiments have shown that they interact and bind efficiently with tubulin protein. Overall, the current study demonstrates that the synthesis of chalcone-amidobenzothiazole conjugates as promising anticancer agents with potent G2/M arrest and apoptotic-inducing activities via targeting tubulin.