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We herein report a convenient radiolabeling method capable of real-time in vivo imaging and biodistribution of bacteria in a living subject. Escherichia coli (E. coli), a model microorganism in this study, was incubated with technetium-99m (99mTc) tricarbonyl under physiological conditions provided radiolabeled bacterial cells. 99mTc-labeled bacterial cells were obtained in high yield (42%, n = 5) and purity (> 99%) within 30 min. In vitro radiolabeling study using cellular components revealed that 99mTc could be incorporated with cytosolic proteins and it was maintained stably in E. coli under several media including mouse serum, high salt solutions with varying pH. SPECT/CT images and organ biodistribution of 99mTc-labeled bacterial cells showed quantitative in vivo behavior and accumulation of E. coli in mice. The radiolabeling method demonstrated here will be a promising platform tool for in vivo tracking and biodistribution of infectious microorganism. [ABSTRACT FROM AUTHOR]