The human topoisomerase IB (hTopoIB) enzyme is a monomeric protein that relaxes the supercoils on double-stranded DNA by forming a covalent DNA/hTopoIB complex by introducing a nick on the DNA strand. Inhibition of hTopoIB results in cell death, which makes this protein a strong target for the treatment of various cancer types, including small-cell lung cancers and ovarian cancers. Camptothecin (CPT) and indenoisoquinoline (IQN) classes of compounds inhibit the hTopoIB activity by intercalating to nicked DNA pairs; however, these inhibitors show different preferences towards DNA bases when bound to the DNA/hTopoIB complex. Here, we investigated the affinities of CPT and one IQN derivative towards different DNA base pairs. The two inhibitors showed different stacking behaviors in the intercalation site and interaction pattern with binding pocket residues, indicating that they have different inhibition mechanisms in the binding pocket that affects the base-pair selectivity. The results obtained from this study are expected to guide researchers in designing gene-specific and more potent compounds to fight cancer through hTopoIB poisoning.