Bacterial infection and biofilm buildup is a major challenge for wound healing. In the present work we developed a method to track bacteria in vivo in order to analyze the progress of the healing. Regulated irrigation combined with negative pressure (RI-NPT) is a nonpharmaceutical therapeutic strategy for reducing bacterial load in acute and chronically infected wounds. Here, we analyzed RI-NPT hydrokinetics and efficacy of bacterial load reduction in wounds. Escherichia coli were loaded with gold nanoparticles, in order to make them "visible" and trackable using computed tomography (CT). Bacteria were tracked using CT in a low-flow rat wound model over time. CT tracking revealed that while regulated negative pressure-assisted wound therapy reduced bacterial load to a limited extent (5%), RI-NPT significantly increased bacterial outflow and clearance (by 45%). This nanotechnology-based approach demonstrates the ability to track bacteria in vivo at real time and also the ability of the RI-NPT technology to effectively reduce bacterial load.