This study evaluates the imaging performance of fluorescent probes which contain the Zn(II)-dipicolylamine (Zn-DPA) affinity ligand. Interaction between the probe and the bacterial cell wall occurs through binding of the anionic phospholipids embedded within the lipid bilayer(s). The targeted phospholipids were identified from titration experiments with liposomes created to serve as a model for bacterial membranes. Treatment of a variety of Gram-negative and Gram-positive strains of bacteria show conclusively that Zn-DPA probes bind to a variety of bacterial pathogens and support the claim that Zn-DPA probes have universal affinity for all strains of bacteria. Molecular probes composed of Zn-DPA ligands linked to a near-infrared fluorophore have utility in detecting bacterial infections in vivo. Imaging studies of murine models infected with Staphylococcus aureus show that bacterial infections can be detected using whole animal imaging equipment within three hours of treatment with the molecular probe. Additional data shows that probe performance is similar in athymic models and mice which contain an active immune system. Clearance of the Zn-DPA probes progresses through the liver, kidneys, and intestines during the 21 hour fluorescent imaging experiment.