This study evaluates the performance of Bis-Zinc II dipicolylamine (ZnII-DPA) conjugated squaraine rotaxane as bacterial targeting probes. Firstly, binding assays with planktonic bacteria show this probe to selectively bind cells best during the log phase of the cell cycle, with up to 99% binding efficiency. These probes binds both Gram-negative and Gram-positive strains of bacteria with varying affinity depending on phospholipid content of Gram-positive cells and structural makeup of the Gram-negative cell wall. Planktonic staining assays and flow cytometry of the amide version of the probe show a threefold increase in the fluorescence intensity when compared to treatments with the triazole version. Phosphatidylserine Sensor 480 analogs with one ZnII-DPA unit exhibit a similar affinity to the bacterial cells, and a faster diffusion rate. Multivalent squaraine rotaxanes exhibit a high affinity for a wide range of bacteria at low concentrations with planktonic bacterial staining assays and slide agglutination assays. Selectivity to the bacteria and cross linking was observed even when treating bacteria in the presence of mammalian cells. Bis-ZnII DPA versions of the probe were more effective as agglutination agents than the mono-ZnII DPA version.