The employment of radiopharmaceuticals is increasing nowadays for infection imaging and early execution of patients having infectious or inflammatory complaints. The main aim of this study was to discover a novel method for the labeling of ofloxacin with (99m)Tc, optimization of labelling conditions to get higher percent yield, to assess kits radiochemical purity, in vitro stability, partition coefficient, protein binding, and intracellular accumulation in Pseudomonas aeruginosa, Salmonella typhi, and Escherichia coli in infected rabbits. Maximum labeling efficiency was achieved when 1.5 mg ofloxacin was labeled with 10-20 mCi sodium pertechnetate in the presence of 3 mg D-penicillamine, 75 μg SnCl₂. In vitro binding and biodistribution in Pseudomonas aeruginosa, Salmonella typhi, and Escherichia coli showed good results. This new complex is efficient for the imaging of infections caused by Gram-positive and Gram-negative bacteria.