New benzothiopyranoindoles (5a-l) and pyridothiopyranoindoles (5m-t), featuring different combinations of substituents (H, Cl, OCH<sub>3</sub>) at R<sup>2</sup>-R<sup>4</sup> positions and protonatable R<sup>1</sup>-dialkylaminoalkyl chains, were synthesized and biologically assayed on three human tumor cell lines, showing significant antiproliferative activity (GI<sub>50</sub> values spanning from 0.31 to 6.93 μM) and pro-apoptotic effect. Linear flow dichroism experiments indicate the ability of both chromophores to form a molecular complex with DNA, following an intercalative mode of binding. All compounds displayed a moderate ability to inhibit the relaxation activity of both topoisomerases I and II, reasonably correlated to their intercalative capacities. Cleavable assay performed with topoisomerase I revealed a significant poisoning effect for compounds 5g, 5h, 5s, and 5t. A theoretical model provided by hydrated docking calculations clarified the role of the R<sup>1</sup>-R<sup>4</sup> substituents on the topoisomerase I poison activity, revealing a crucial role of the R<sup>2</sup>-OCH<sub>3</sub> group.