A series of novel 5-(substituted phenyl)-3-[(substituted phenylamino)methyl]-3H-[1,3,4]oxadiazole-2- thione derivatives were prepared and their in vitro cytotoxicity was evaluated against a panel of three cancer cell lines, namely, hepatocarcinoma HepG2, breast adenocarcinoma MCF-7, and leukemia HL-60 cells, using the widely accepted MTT assay. In general, the synthesized compounds displayed weak to moderate cytotoxic activity against the three tested cell lines. Compound 5a, which has trimethoxy substituents on both phenyl rings, exhibited the highest cytotoxic effect against all cell lines tested with IC50 values of 12.01, 7.52 and 9.7 μM against HepG2, MCF-7 and HL-60 cells, respectively. Mechanistic studies revealed that the test compounds showed a good inhibitory effect on cellular tubulin of hepatocellular carcinoma. Compound 5h was the most potent tubulin inhibitor in HepG2 cells, with 81.1 % inhibition of the original control tubulin. Moreover, the mechanism of tubulin polymerization inhibition was confirmed by immunofluorescence assay, flow cytometry, and docking study.