Microbial detection is crucial for the control and prevention of infectious diseases, being one of the leading causes of mortality worldwide. Among the techniques developed for bacterial detection, those based on metabolic indicators are progressively gaining interest due to their simplicity, adaptability, and, most importantly, their capacity to differentiate between live and dead bacteria. Prussian blue (PB) may act as a metabolic indicator, being reduced by bacterial metabolism, producing a visible color change from blue to colorless. This molecule can be present in two main forms, namely, the soluble and the insoluble, having different properties and structures. In the current work, the bacterial-sensing capacity of soluble and insoluble PB will be tested and compared both in suspensions as PB-NPs and after deposition on transparent indium tin oxide-poly(ethylene terephthalate) (ITO-PET) electrodes. In the presence of live bacteria, PB-NPs are metabolized and completely reduced to the Prussian white state in less than 10 h for soluble and insoluble forms. However, when electrodeposited on ITO-PET substrates, less than 1 h of incubation with bacteria is required for both forms, although the soluble one presents faster metabolic reduction kinetics. This study paves the way to the use of Prussian blue as a metabolic indicator for the early detection of bacterial infection in fields like microbial diagnostics, surface sterilization, food and beverage contamination, and environmental pollution, among others.