A series of 2-phenylamino-3-acyl-1,4-naphtoquinones were evaluated regarding their in vitro antiproliferative activities using DU-145, MCF-7 and T24 cancer cells. Such activities were discussed in terms of molecular descriptors such as half-wave potentials, hydrophobicity and molar refractivity. Compounds4and11displayed the highest antiproliferative activity against the three cancer cells and were therefore further investigated. The in silico prediction of drug likeness, using pkCSM and SwissADME explorer online, shows that compound11is a suitable lead molecule to be developed. Moreover, the expressions of key genes were studied in DU-145 cancer cells. They include genes involved in apoptosis (Bcl-2), tumor metabolism regulation (mTOR), redox homeostasis (GSR), cell cycle regulation (CDC25A), cell cycle progression (TP53), epigenetic (HDAC4), cell-cell communication (CCN2) and inflammatory pathways (TNF). Compound11displays an interesting profile because among these genes,mTORwas significantly less expressed as compared to control conditions. Molecular docking shows that compound11has good affinity with mTOR, unraveling a potential inhibitory effect on this protein. Due to the key role of mTOR on tumor metabolism, we suggest that impaired DU-145 cells proliferation by compound11is caused by a reducedmTORexpression (less mTOR protein) and inhibitory activity on mTOR protein.