Bacterial infection is one of the vital reasons of morbidity and mortality, especially in developing countries. It appears silently without bothering the geological borders and imposes a grave threat to humanity. Nuclear medicine technique has an important role in helping early diagnosis of deep-seated infections. The aim of this study was to develop a new radiopharmaceutical Tc-99m-labeling sulfadiazine as an infection imaging agent. Radiolabeling of sulfadiazine with technetium-99m (Tc-99m) was carried out using stannous tartrate as a reducing agent in the presence of gentistic acid at pH = 5. The quality control tests revealed similar to 98% labeling efficiency. Paper chromatographic (PC) and instant thin-layer chromatographic (ITLC) techniques were used to analyze radiochemical yield. Biodistribution and infection specificity of the radiotracer were performed with Escherichia coli (E. coli) infection-induced rats. Scintigraphy and glomerular filtration rate (GFR) study was performed in E. coli-infected rabbits. Scintigraphy indicated E. coli infection targeting potential of Tc-99m-SDZ, while biodistribution study showed minimal uptake of Tc-99m-SDZ in non-targeted tissues. The uptake in the kidneys was found 2.56 +/- 0.06, 2.09 +/- 0.10, and 1.68 +/- 0.09% at 30 min, 1 h, and 4 h, respectively. The infected muscle (target) to non-infected muscle (non-target) ratio (T/NT) was found 4.49 +/- 0.04, 6.78 +/- 0.07, and 5.59 +/- 0.08 at 30 min, 1 h, and 4 h, respectively.