Colorectal cancer is a global burden, today. Currently, the drop in the number of drugs approved by FDA and their limited success, enforce medicinal chemists and biotech industries to develop novel drugs to meet the demands of the patient population. In the present research study, we synthesized six novel compounds of methoxy substituted (Z)‒5‒Benzylidene‒1,3‒thiazolidine‒2,4‒dione via Knoevenagel condensation reaction. All the newly synthesized compounds were tested for their cytotoxic effect. Cytotoxic evaluation was carried out by using MTT reagent on viable human colorectal cancer cell lines: SW48, HT29 and Caco2 and normal colon cell line, CCD 841 CoN. The IC50 values were determined for tested compounds revealing cytotoxic activity. Among all the tested analogues, compound C#06 revealed highest potency on cell line SW48 with an IC50 value of 10.34 ± 2.25. Therefore, it may be implied that the novel compound C#06 appears to be a very promising agent for colorectal cancer treatment.