In the present study, 1-(un)substituted 2-(hetero)aryl imidazo[4,5-b]phenazines 4a-j and 6a-d were synthesized and evaluated for their cytotoxic activities against a panel of cell lines at 10 micromolar concentration. Compound 4f revealed a remarkable and broad spectrum of cytotoxic activity with growth inhibition percent (GI%) of 11-82%. It was found that cell lines derived from leukemia, and breast cancer were the most sensitive to the imidazophenazine derivative 4f. It showed GI% of 82% against MOLT-4 cell line from leukemia. Moreover, compound 4e showed GI% of 88% against SK-OV-3 cells from ovarian cancer. In addition, compound 4b showed GI% of 51% against M14 melanoma cell line, whereas compound 6a showed GI% of 44% against T-47D breast cancer cell line. The most promising compounds 4a and 4e-g were further tested for their Topo I and Topo IIα inhibitory activities. It was found that compound 4e is the most potent derivative against Topo I in comparison to camptothecin (IC50 = 29.25 and 25.71 µM, respectively), whereas the imidazophenazine derivatives 4f and 4g displayed comparable potency to etoposide against Topo IIα (IC50 = 26.74, 22.72, and 20.52 µM, respectively). Investigation of the effect of compound 4f on MCF-7 cell cycle at its IC50 concentration showed its effectiveness in arresting the cell cycle at the G2/M phase; furthermore, it induced apoptosis in MCF-7 cells. Molecular docking simulations in Topo I and Topo IIα revealed that the biological activity of the target compounds could be due to their mechanism of action that resembles the topoisomerase poisons which involves the accommodation of their polycyclic scaffold in the DNA cleavage site stacking between the base pairs interacting through several π-π stacking interactions with the surrounding DNA bases stabilizing the topoisomerase/DNA cleavage complex preventing the re-ligation reaction. SwissADME web tool proved that compounds 4f and 4g exhibit promising ADME profile, and drug likeness properties.