To seek a novel 99m Tc-labeled quinolone derivative for bacterial infection SPECT imaging that aims to lower nontarget organ uptake, a novel norfloxacin 6-hydrazinoicotinamide (HYNIC) derivative (HYNICNF) was designed and synthesized. It was radiolabeled with different coligands, such as tricine, trisodium triphenylphosphine-3,3′,3″-trisulfonate (TPPTS), sodium triphenylphosphine-3-monosulfonate (TPPMS), and ethylenediamine-N,N′-diacetic acid (EDDA), to obtain three 99m Tclabeled norfloxacin HYNIC complexes, namely, [ 99m Tc]Tc-tricine-TPPTS-HYNICNF, [ 99m Tc]Tc-tricine-TPPMS-HYNICNF, and [ 99m Tc]Tc-EDDA-HYNICNF. These complexes were purified (RCP > 95%) and evaluated in vitro and in vivo for targeting bacteria. All three complexes are hydrophilic, maintain good stability, and specifically bind Staphylococcus aureus in vitro. The biodistribution in mice with bacterial infection demonstrated that [ 99m Tc]Tc-EDDA-HYNICNF showed a higher abscess uptake and lower nontarget organ uptake and was able to distinguish bacterial infection and sterile inflammation. Single photon emission computed tomography (SPECT) image study in bacterial infection mice showed there was a visible accumulation in the infection site, suggesting that [ 99m Tc]Tc-EDDA-HYNICNF is a potential radiotracer for bacterial infection imaging.