A series of four new, cyclometallated ruthenium (II) complexes was synthesized from 3-acetyl-8-methoxy-2H-chromen-2-one functionalized 4(N)- substituted thiosemicarbazones and characterized through various spectral and analytical methods. The molecular structures of the complexes 1, 2 and 4 were determined by single-crystal X-ray diffraction analysis, which confirmed that the complexes possess a distorted octahedral geometry with the ligands coordinating in a dibasic tridentate fashion via C, N and S atoms. DNA Calf Thymus DNA (CT-DNA) and protein Bovine Serum Albumin (BSA) and Human Serum Albumin (HSA) binding studies indicated an intercalative mode of binding with DNA and static quenching mechanism with proteins. The compounds cleaved plasmid DNA (pBR322) without application of any external agent and acted well as free radical scavengers. A good spectrum of antimicrobial activity was observed against four bacterial and five fungal pathogens. Two cancerous cell lines, MCF-7 (human breast cancer) and A549 (human lung carcinoma) were employed to test their in vitro cytotoxic activity using MTT assay. The complexes (1–4) showed better activity with lower IC50 values over cisplatin. Further non toxic nature of the complexes have been examined with human normal keratinocyte cell line HaCaT. Further, the results of Lactate dehydrogenase release (LDH) and Nitric Oxide (NO) release supported the cytotoxic nature of the compounds. The results of all the biological studies carried out implied that the complex 3 bearing an ethyl substituent was observed to be the best. Display omitted •Four new organoruthenium (II) complexes have been synthesized and characterized.•The DNA/protein interactions of ligands and complexes were studied by a variety of techniques.•The antimicrobial activity against four different bacteria and five fungi have also been examined.•The antiproliferative activity was evaluated against MCF-7 and HeLa cell lines.•The complexes 1–4 showed potent anticancer activity over the standard drug, Cisplatin.