Indole thiosemicarbazones (TSCs) and their complexes are known to possess various biological activities. The variation in anticancer activity with different indole-N substituents of TSCs in the RuII-benzene complexes (C1-C7) was studied. The complexes were adequately characterized using analytical and spectroscopic techniques. The single crystal X-ray diffraction (XRD) technique confirmed the piano-stool structure of the complexes (C4 and C7). The theoretical findings on the structure of complexes supported the experimental results. The complexes (C1-C7) exhibited good biomolecular interactions with DNA/protein and significant anticancer potential against MB-MDA-231 and MCF-7 cancer cells. Also, the complexes were least toxic to normal human cells, suggesting the selectivity of the complexes. The benzyl substituent at indole-N of the TSC ligands seemed to improve the cytotoxic profile of their complexes compared to the allyl one. Among the benzyl scaffolds, the para-substituted [methyl (C5) and chloro (C6)] ones elevated the anticancer activity compared to the meta-substituted compounds (C4 and C7). Hoechst and AO-EB staining assisted the visualization of the apoptotic changes induced by active complexes C2 and C6 in MB-MDA-231 cells. Further, flow cytometric analysis authenticated the cell cycle arrest in the sub-G0/G1 phase. Western blotting studies confirmed the apoptotic mode of cell death by quantifying the proapoptotic and antiapoptotic proteins.