Thiazole and its derivatives have received a lot of attention from researchers due to its wide biological, pharmacological, and anticancer properties. A novel series of 2-[2-[4-Hydroxy-3-substituted benzylidene hydrazinyl]-thiazole-4[5H]-ones (4a–c) and acetoxy derivative (5) were synthesized via using thiosemicarbazones (2a–c). The structure of the thiazole derivatives (4a–c) and 5 in these compounds was confirmed by spectroscopic techniques (IR and NMR), as well as elemental investigations. The synthesized derivatives biological activity was assessed based on their capacity to suppress the growth of the cancer cell lines MCF-7 and HepG2, as well as to halt the cell cycle and trigger apoptosis. Among the synthesized thiazole derivatives, compound 4c was found the most active derivative, with inhibitory concentrations IC₅₀ = 2.57 ± 0.16 and 7.26 ± 0.44 µM in MCF-7 and HepG2, respectively, compared to Staurosporine as the standard drug with IC₅₀ 6.77 ± 0.41 and 8.4 ± 0.51 µM, respectively. Additionally, compound 4c blocked vesicular endothelial growth factor receptor-2 (VEGFR-2), according to our results (IC₅₀ = 0.15 µM), compared to Sorafenib (IC₅₀ = 0.059 µM) as the standard drug. Moreover, compound 4c induced cell cycle arrest at the G1/S phase, increasing the percentage and accumulation of cancer cells (DNA content) in the pre-G1 phase by 37.36% in MCF-7 cancer cells compared to untreated MCF-7 cells at 2.02%. Also, compound 4c increased the percentage of early and late apoptosis from 0.51% and 0.29%, respectively, in the case of the MCF-7 untreated control sample to 22.39% and 9.51%, respectively, in the MCF-7 treated sample. Furthermore, molecular docking studies of compounds 4a–c and 5 were conducted with four key proteins (aromatase, epidermal growth factor receptor (EGFR), cyclin-dependent kinase 2 (CDK2), and B-cell lymphoma 2 (Bcl-2)) that stimulate the growth, proliferation, and development of cancer cells. Compound 4c exhibited good docking scores with a promising and potential binding affinity toward the active site of selected docked proteins. According to these results, compound 4c showed efficient cytotoxic activity against the tested cancer cells.