A series of non-symmetric dinuclear polypyridylruthenium(ii) complexes (Rubbn-Cl) that contain one inert metal centre and one coordinatively-labile metal centre, linked by the bis[4(4′-methyl-2,2′-bipyridyl)]-1,n-alkane ligand ("bbn" for n = 7, 12 and 16), have been synthesised and their potential as antimicrobial agents examined. The minimum inhibitory concentrations (MIC) of the ruthenium(ii) complexes were determined against four strains of bacteria - Gram-positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). The Rubbn-Cl complexes displayed good antimicrobial activity, with Rubb12-Cl being the most active complex against both Gram-positive and Gram-negative strains. Interestingly, Rubb7-Cl was found to be eight- and sixteen-fold more active towards E. coli than against S. aureus and MRSA, respectively. The cytotoxicities of the Rubbn-Cl complexes against three eukaryotic cell lines-two kidney cell lines (BHK and HEK-293) and one liver cell line (HepG2)-were examined. The Rubbn-Cl complexes were found to be considerably less toxic towards eukaryotic cells than S. aureus, MRSA and E. coli, with Rubb12-Cl being thirty- to eighty-times more toxic to the bacteria than to BHK, HEK-293 or HepG2 cells. Unexpectedly, Rubb7-Cl was far more toxic to HepG2 cells (24 h-IC50 = 3.7 μM) and far less toxic to BHK cells (24 h-IC50 = 238 μM) than the Rubb12-Cl and Rubb16-Cl complexes. In order to understand the unexpected large differences in the cytotoxicities of the Rubbn-Cl complexes towards eukaryotic cells, a confocal microscopic study of their intracellular localisation was undertaken. The results suggest that the observed cytotoxicity might be related to the extent of DNA binding.