In the present study, new Schiff bases derived from pentafluorophenyl-hydrazine (L1, L2, L3, L4) were synthesized and their structures were characterized by 1 H nuclear magnetic resonance spectroscopy (NMR), 13 C NMR, 19 F NMR, fourier transform infrared spectroscopy, and elemental analysis methods. Then, the anticancer activities of the obtained compounds were investigated using three human cancer cell lines (A2780, over; Caco-2, colon; and HT-29 colon carcinoma cell lines). According to the obtained cytotoxicity results, compound number L4 was found to have the highest anticancer activity in A2780 (over) and Caco-2 (colon) cell lines. Furthermore, in silico, ADMET properties, where studies play an important role in the development and prediction of drug compounds, were calculated using web-based platforms. In addition, molecular docking studies were performed to evaluate the binding interactions between the synthesized pentafluorophenyl-hydrazone compounds and the MDM2 protein (4JSC). Both in vitro and in silico results showed that the synthesized compounds could act as potent anticancer agents.