Ruthenium(II) complexes containing the tetradentate ligand bis[4(4′-methyl-2,2′-bipyridyl)]-1,n-alkane (“bbn”; n=10 and 12) have been synthesised and their geometric isomers separated. All [Ru(phen)(bbn)]2+ (phen=1,10-phenanthroline) complexes exhibited excellent activity against Gram-positive bacteria, but only the cis-α-[Ru(phen)(bb12)]2+ species showed good activity against Gram-negative species. In particular, the cis-α-[Ru(phen)(bb12)]2+ complex was two to four times more active than the cis-β-[Ru(phen)(bb12)]2+ complex against the Gram-negative strains. The cis-α- and cis-β-[Ru(phen)(bb12)]2+ complexes readily accumulated in the bacteria but, significantly, showed the highest level of uptake in Pseudomonas aeruginosa. Furthermore, the accumulation of the cis-α- and cis-β-[Ru(phen)(bb12)]2+ complexes in P. aeruginosa was considerably greater than in Escherichia coli. The uptake of the cis-α-[Ru(phen)(bb12)]2+ complex into live P. aeruginosa was confirmed by using fluorescence microscopy. The water/octanol partition coefficients (log P) were determined to gain understanding of the relative cellular uptake. The cis-α- and cis-β-[Ru(phen)(bbn)]2+ complexes exhibited relatively strong binding to DNA (Kb≈106 M−1), but no significant difference between the geometric isomers was observed.