Three new ruthenium(II)-arene complexes, Ru(η6-p-cymene)(L1)Cl2 (C1) where L1 is N-((4 methoxyphenyl)carbamothioyl)benzamide; Ru(η6-p-cymene)(L2)Cl2 (C2) where L2 is 4-(3-benzoylthioureido)benzoic acid and Ru(η6-p-cymene)(L3)Cl2 (C3) where L3 is methyl 4-(3- benzoylthioureido)benzoate have been synthetized, characterized and evaluated for their antimicrobial and anticancer activity. Characterization was performed using 1H and 13C NMR, IR spectroscopy, mass spectrometry, electrical conductivity measurements and X-Ray diffraction analysis. X-Ray diffraction analysis of C1 showed typical expected “piano-stool” geometry with ruthenium coordinated to ligand via nitrogen and sulfur atoms of benzoylthiourea derivatives. Interesting, in herein described complex, upon coordination the four-membered ring was formed, instead of six-membered chelate common for this type of ligands. Cytotoxic activity was determined in human cervix adenocarcinoma (HeLa) cell line and IC50 values ranged from 29.68 to 52.36 μM and the complexes were more active than related ligands (except in case of C2 where it is found that IC50 value is close to IC50 value of related ligand). Complex Ru(η6-p-cymene)(L1)Cl2 (C1) expressed the highest cytotoxic activity with IC50 value of 29.7 μM. Complexes and ligands were tested against nine Gram-positive and Gram-negative bacteria and one yeast- Candida albicans. Clinical Candida spp. strains from microbiological laboratories were included in testing processes as well. Minimum inhibitory concentrations values ranged from 62.5 μg/ml for complexes against Candida albicans to over 1000 μg/ml for several bacterial species. Three new ruthenium(II)-arene complexes with benzoylthiourea derivatives as ligands have been synthetized, characterized and evaluated for their antimicrobial and anticancer activity. Antimicrobial activity has been evaluated against nine Gram-positive and Gram-negative bacterial strains and one yeast, and anticancer activity against human cervix adenocarcinoma (HeLa) cell line. Display omitted •Three new organometallic Ru(II) complexes were synthesized and characterized.•Coordination via the four-membered ring of the ligand was determined with X-Ray analysis.•Cytotoxic activity was determined in human cervix adenocarcinoma (HeLa) cell line.•Antimicrobial activity was tested against nine gram-positive and gram-negative bacteria.•Antimicrobial activity was also tested against one yeast.