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Background: Radiotracers techniques are offering a unique way to diagnosis deep tissue infection in its early stages. The radiotracers including radio-antibiotics have shown promising results in the early diagnosis of infection and its discrimination from infectious foci but wide ranges of microorganisms still poses threats., Objectives: Synthesis of Technetium-99m ((99m)Tc)-temafloxacin (TMC) complex for the localization of in vivo Streptococci pneumoniae infection in the early stages., Material and Methods: The (99m)Tc-TMC complex was prepared by adding 50 µg of stannous chloride (SnCl2) with 37 MBq (0.5 mL) of sodium pertechnetate (Na(99m)TcO4-) at a pH of 5.2. Then 1 mg of the TMC was added to the mixture followed by incubation at room temperature for 10 min. The same procedure was repeated by changing the amount of the SnCl2 from 50 to 250 µg along with the TMC from 2 to 5 mg and Na(99m)TcO4- from 74 to 185 MBq. In higher concentrations of cysteine the stability of the (99m)Tc-TMC complex was evaluated. In vitro Streptococci pneumoniae uptake was investigated to validate the accuracy and preciseness of the (99m)Tc-TMC complex. In vivo uptake of the (99m)Tc-TMC complex was evaluated in ten (10) normal male Wistar rats (MWR) (140-160 g) divided into two groups (I and II)., Results: Maximum stability of 98.00 ± 0.34% at 30 min after reconstitution was observed by mixing 2.5 mg of TMC, 100 µg of SnCl2 with 74 MBq of the Na(99m)TcO4-. The stability of the complex remained 90% up to 4 hours. In serum the complex showed stability up to 16 hours. A saturated in vitro binding was noted with live Streptococci pneumoniae. In the infected region (left thigh) of the MWR, almost five times higher uptake was observed as compared to the inflamed and normal muscles., Conclusions: The above results confirm the suitability of the (99m)Tc-TMC complex as a potential Streptococci pneumoniae infection localizing agent.