Dinuclear polypyridylruthenium(II) complexes bridged by a flexible methylene linker have received considerable interest as potential antibacterial agents. Their potency and uptake into bacterial cells is directly modulated by the length of the bridging linker, which has implicated membrane interactions as an essential feature of their mechanism of action. In this work, a combination of molecular dynamics (MD) simulations and solid-state NMR was used to present an atomistic model of a polypyridylruthenium(II) complex bound and incorporated into a bacterial membrane model. The results of P-31, H-2, H-1, and C-13 NMR studies revealed that the antibacterial [{Ru(phen)(2)}(2)(mu-bb(12))](44) complex (Rubb12), where phen = 1,10-phenanthroline and bb12 = bis[4(4'-methyl-2,2'-bipyridy1)]-1,12-dodecane), incorporated into a negatively charged model bacterial membrane, but only associated with the surface of a charge-neutral model of a eukaryotic membrane. Furthermore, an inactive [{Ir(phen)2}2(p-bb12)]6+ (Irbbn) analogue, which is not taken up by bacterial cells, maintained only a surface-bound association with both bacterial and eukaryotic model membranes according to P-31 and H-2 NMR The effects of Rubb12 on 31P chemical shift anisotropy and 2H acyl chain order parameters for negatively charged membranes correlated with a membrane -spanning state of the complex according to MD simulation in which the metal centers embed in the lipid head group region and the central void, created by the biconic shape of the complex, resulting in increasing disorder of lipid acyl chains and membrane -thinning. A transbilayer mechanism and membrane -spanning may be essential for the cellular uptake and antibacterial activity of this class of compounds.