A series of fluorinated 3,6-diaryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles (2a–2i) was synthesized by condensation of various substituted 4-amino-5-phenyl-4H-1,2,4-triazole-3-thiols (1a–1i) with penta fluoro benzoic acid in good yields (60–80%). The synthesized compounds were screened for anticancer activity against three cancerous cell lines MCF7 (human breast cancer), SaOS-2 (human osteosarcoma) and K562 (human myeloid leukemia). The compounds showed moderate to good antiproliferative potency against the studied cell lines. Among these, compound 2b showed higher antiproliferative activity (IC50 22.1, 19 and 15 μM against MCF7, SaOS-2 and K562, respectively) while 2a exhibited least antiproliferative activity (IC50 30.2, 39 and 29.4 μM against MCF7, SaOS-2 and K562 cells, respectively). Therefore, the present study demonstrates that fluorine substituted 3,6-diaryl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazoles would be a better prospective in the development of anticancer drugs.