In this study femtosecond and nanosecond time-resolved transient absorption spectroscopy was used to investigate the influence of ionic strength and complexity on the excited state dynamics of a Ru(II)-based metal-organic dyad. The bis-heteroleptic complex [Ru(bpy)2(ippy)](2+) (1), where bpy = 2,2 '-bipyridine and ippy = 2-(1-p-yrenyl-1H-imidazo[4,5-f][1,10]phenanthroline, is a potent photosensitizer for in vitro photodynamic therapy (PDT) and photodynamic inactivation (PDI) of microorganisms owing to a long-lived triplet excited state derived from a metal-to-ligand charge-transfer ((MLCT)-M-3) state that is equilibrium with an intraligand ((IL)-I-3) state. The prolonged lifetime provides ample opportunity for bimolecular quenching of this state by oxygen; thus singlet oxygen (O-1(2)) sensitization is very efficient. In simple aqueous solution, fast cooling within the (MLCT)-M-3 manifold is followed by energy transfer to an (IL)-I-3 state, which is facilitated by rotation of a pyrenyl unit about the imidazo-pyrenyl (ip) coannular bond. For solutions of 1 in high ionic strength simulated biological fluid (SBF), a more physiologically relevant solvent that contains a complex mixture of ions at pH 7.4, femtosecond studies revealed an additional excited state, possibly based on an ion-ligand interaction. This new state appearing in high ionic strength SBF was not observable in water, simple buffers, or low ionic strength SBF. These photoinduced dynamics were also affected by the presence of biomolecules such as DNA in simple buffer, whereby relaxation on the picosecond time scale was accelerated from 39 to 18 ps with DNA intercalation by 1. The increased rate of coplanarization of the pyrene and the imidazole units was attributed to DNA-induced conformational restriction of the pyrenyl unit relative to the ip bond. Quantitative changes to excited state decay rates of 1 in solutions of high ionic strength were also observed when probed on the microsecond time scale. Notably, the thermalized excited state decay pathways were altered substantially with DNA intercalation, with access to some states being completely blocked. Experimentally, this manifested in the absence of the slowest microsecond decay channel, which is normally observed for 1 in solution. The quantitative and qualitative observations from this study highlight the importance of employing biologically relevant solvents and potential biomolecule targets when the excited state dynamics and photophysical properties (under cell-free conditions) responsible for the potent photobiological effects are assessed in the context of photodynamic therapy and photodynamic inactivation.