The antitumor activity of the newly synthesized 5-arylidenethiazolidine-2,4-dione derivatives 18a–f and 19a–f was investigated, compared to doxorubicin (IC50 = 4.17–8.87 μM) and SAHA (IC50 = 2.70–7.11 μM). Among the tested molecules, compounds 18b, 18c, 18f, 19d, and 19e displayed the highest antitumor activity against cancer cell lines (IC50 = 3.16–28.94 μM). Further, compounds 18b, 18c, 18f, and 19d were tested as Histone deacetylases (HDACs) inhibitors compared with Entinostat (IC50 = 0.093–0.75 μM). Compounds 18b, 18c, 18f, and 19d inhibited HDAC1, HDAC2, HDAC8, and HDAC6 enzymes with IC50 values ranging from 0.144 to 1.741 μM. In addition, compound 18b caused apoptosis via a mitochondrial-mediated pathway and led to cell cycle arrest at the G1 phase. It also increased caspases-3 and caspases-7 by 5.2–3.9 and 9.1–3.7 folds, respectively. The molecular docking analysis of compounds 18b and 18c revealed that they could bind to the active sites of HDAC1, HDAC2, HDAC8, and HDAC6 like co-crystallized inhibitors.
