New series of isoxazole derivatives were synthesized and evaluated for in vitro antitumor activity against HepG2, MCF-7 and HCT-116 cancer cells. Results showed that 4b and 25a are the most potent members against the three cancer cells (IC = 6.38-9.96 μM). Further, 4a, 8a and 16b showed strong activity against the three cancer cells, whereas 6b, 10a, 10b and 16a exhibited moderate activity against the three cancer cells. Moreover, 25a showed low cytotoxicity against WISH and WI38 normal cells (IC = 53.19 ± 3.1 and 38.64 ± 2.8 µM, respectively), and it might be used as a potent and safe antitumor agent. The nine active compounds 4a, 4b, 6b, 8a, 10a, 10b, 16a, 16b and 25a were studied for EGFR-TK inhibitory activity, where 10a, 10b and 25a showed the highest inhibitory activity (IC = 0.064 ± 0.001, 0.066 ± 0.001 and 0.054 ± 0.001 µM, respectively). Compound 25a was also assessed against other four target proteins, and it showed promising inhibitory activities against VEGFR-2, CK2α and topoisomerase IIβ, and acceptable inhibitory activity against tubulin polymerization. Cell cycle analysis of cancer cells treated with 25a proved that it induces cell cycle arrest at G2/M and pre-G1 phases. Furthermore, it was confirmed that 25a induces cancer cell death through apoptosis, supported by increased caspases 3/9 levels and increased Bax/Bcl-2 ratio in the three cancer cells. In addition, docking studies proved the exact fit of 25a into the active site of EGFR-TK, VEGFR-2, CK2α, topoisomerase IIβ and tubulin. Lipinski's rule and Veber's standards were also analyzed, and results illustrated that 25a is expected to be well absorbed orally.