Bacterial infection poses life-threatening challenge to humanity and stimulates to the researchers for developing better diagnostic and therapeutic agents complying with existing theranostic techniques. Nuclear medicine technique helps to visualize hard-to-diagnose deep-seated bacterial infections using radionuclide-labeled tracer agents. Metronidazole is an antiprotozoal antibiotic that serves as a preeminent anaerobic chemotherapeutic agent. The aim of this study was to develop technetium-99m-labeled metronidazole radiotracer for the detection of deep-seated bacterial infections. Radiosynthesis of Tc-99m-metronidazole was carried by reacting reduced technetium-99m and metronidazole at neutral pH for 30 min. The stannous chloride dihydrate was used as the reducing agent. At optimum radiolabeling conditions, similar to 94% radiochemical was obtained. Quality control analysis was carried out with a chromatographic paper and instant thin-layer chromatographic analysis. The biodistribution study of radiochemical was performed using Escherichia coli bacterial infection-induced rat model. The scintigraphic study was performed using E. coli bacterial infection-induced rabbit model. The results showed promising accumulation at the site of infection and its rapid clearance from the body. The tracer showed target-to-non-target ratio 5.57 +/- 0.04 at 1 h post-injection. The results showed that Tc-99m-MNZ has promising potential to accumulate at E. coli bacterial infection that can be used for E. coli infection imaging.