In our pioneering seminal work on novel cytotoxic potential agents, we are detailing the virtual screening-guided synthesis, characterization, and cytotoxic evaluation of Bis(9-acridinyl) ketone derivatives, specifically Novel N10-substituted 2-fluoro-7-methyl and 2,7-dimethyl Bis-Acridone derivatives. The study substituted Bis-acridone derivatives were investigated as topoisomerase inhibitors. Molecular docking studies against 1TL8 (Topoisomerase I) and 3QX3 (Topoisomerase IIβ) proteins revealed promising ligand–protein binding interactions among 16 compounds, with four showing high binding affinity for Topoisomerase IIβ (3QX3). Compounds BFM, BPL, BPR, and BBT exhibited the highest binding affinities, with BFM and BBT displaying notable significant in-vitro cytotoxic effects were observed against Human Breast Adenocarcinoma (MCF-7) cell lines, with IC50 values recorded at 23 ± 1.8 µg and 31 ± 2.2 µg. Furthermore, molecular dynamics simulations validated the stability of these compounds. The in-silico ADME analysis indicated that compound BPL has the potential to penetrate the blood-brain barrier (BBB), while all synthesized compounds were evaluated accordingly demonstrated favorable bioavailability and low toxicity profiles, positioning them as promising candidates for further optimization as topoisomerase inhibitors in cancer therapy.