Benzo[d][1,3]dioxoles 1,4-thiazepines remarkable antitumor activities, benzo[d][1,3]dioxoles-fused 1,4-thiazepines, which combine two biologically active heterocyclic cores, are expected to be of pharmacological interest, We therefore envisaged that integrating 1,4-thiazepine and benzo[d][1,3]dioxole moieties in one molecular platform could potentially produce novel compounds with significant synergistic antitumor properties. A series of novel benzo[d][1,3]dioxoles-fused 1,4-thiazepines, designed via molecular hybridization approach, were synthesized in very good yields using one-pot condensation of 3,4-methylenedioxyaniline, aldehydes, and α-mercaptocarboxylic acids under solvent-free condition. The anti-proliferative activities of all the synthesized compounds were assessed on two different human cancer cell lines (Esophageal squamous cell carcinoma Ec9706 and Eca109), and the results showed that compound 4e showed the best anti-tumor activity with IC50 values of 8.23 μM and 16.22 μM against Ec9706 and Eca109 cell lines, respectively, which was 2-3 times more potent than 5-Fluorouracil (IC50 = 23.26 μM and 30.25 μM against Ec9706 and Eca109 respectively). These novel benzo[d][1,3]dioxoles fused with bioactive heterocyclic skeletons may find their pharmaceutical applications after further investigations.