Tc-99m-ciprofloxacin scintigraphy is useful in the detection of gram-positive and gram-negative bacterial infections and also for differentiating the infection from aseptic inflammation. However, due to growing bacterial resistance to antibiotics, the Tc-99m-ciprofloxacin no longer can be effective in broad-spectrum infection imaging as it is gradually losing specificity. In this study, we are presenting our findings regarding the in vitro and in vivo susceptibility of Tc-99m-ciprofloxacin for multi-drug-resistant Staphylococcus aurous, Escherichia coli, and Pseudomonas aeruginosa bacterial strains which were isolated from clinical samples. The results of radiosynthesis of Tc-99m-ciprofloxacin showed more the 95% radiochemical purity and less than 5% radioactive impurities. In vitro Tc-99m-ciprofloxacin susceptibility test showed that E. coli offered more resistant to Tc-99m-ciprofloxacin as compared to S. aurous and P. aeruginosa. In vivo study using bacterial infection induced rabbit model also revealed lowest uptake by E. coli lesion. The T/NT values were obtained 1.96 +/- 0.15 in the case of E. coli; 2.81 +/- 0.51 in the case of S. aurous; and 2.32 +/- 0.66 in the case of P. aeruginosa at 4h post-injection. The SPECT infection imaging of S. aurous, E. coli, and P. aeruginosa bacterial infection induced rabbit models also indicated the clear accumulation in S. aurous and P. aeruginosa lesions while negligible uptake by E. coli lesion further verify the in vitro and in vivo susceptibility profile. On the bases of the results obtained, the Tc-99m-ciprofloxacin showed selective and poor broad spectrum SPECT infection imaging.