A series of S-alkyl derivatives of 3-(substituted-(1,1′-biphenyl)-3-yl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole-6-thiol (4a–j) were synthesized by conventional and microwave irradiation methods. Microwave method proved to be a rapid and better yield compare to that of conventional method. The synthesized compounds were characterized by 1H NMR, 13C NMR, 19F NMR, IR, LCMS, and elemental spectroscopic analysis. These compounds were screened for their in vitro anticancer activity by MTT assay. Among the tested compounds, the compound 4c was the most promising anticancer agent with IC50 value 12 μM in HT29 cell line.