Leveraging their potential anticancer properties, two novel series of quinazolinone-based scaffolds, 3a-i and 7a-i, have been designed, synthesized, and scanned for their anticancer efficacy across three diverse human cancer cell lines, HepG-2, MCF-7, and HCT-116, alongside a normal cell line (BJ-1). Erlotinib and Doxorubicin served as the reference drugs. Notably, derivatives 3i and 7f exhibited the most potent activity against HepG-2, with IC50 values of 1.66 μM and 1.67 μM, respectively, demonstrating about two-fold greater potency than erlotinib and doxorubicin (IC50 = 2.85 μM and 4.25 μM, respectively). Additionally, compound 7i showed superior efficacy against MCF-7 with an IC50 of 3.25 μM, outperforming erlotinib and doxorubicin (IC50 = 3.56 μM and 5.38 μM, respectively). In the case of colon cancer (HCT-116), compound 7i also displayed the highest cytotoxic activity compared to erlotinib and doxorubicin (IC50 = 1.20 μM versus 3.05 and 5.70 μM, respectively). Notably, most tested compounds exhibited a favorable safety profile against the normal human cell line (BJ-1). Furthermore, the derivatives demonstrated significant inhibitory properties on the Epidermal Growth Factor Receptor (EGFR) besides its mutations, EGFRL858R and EGFRT790M, compared with Erlotinib, the reference drug. Compound 7f notably increased Bax and Bcl-2 levels by 1.9 and 1.3 folds, respectively, relative to Erlotinib. Moreover, 7f induced the apoptotic effect, arrested the cell cycle at the G0/G1 phase, and halted the mitotic cycle in HepG-2 cells. To further validate these findings, docking simulations of the promising derivatives 7i and 7f were conducted to assess their anticipated binding affinities with EGFR and its T790M/L858R mutants. Thus, compound 7f has the potential to be developed into a potent anticancer agent.