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A simple methodology was developed to evaluate binding efficiency of antibiotic members of fluoroquinolones, namely ciprofloxacin, ofloxacin and enorfloxacin, complexed with 99m Tc, against Escherichia coli , Salmonella and Pseudomonas aeruginosa bacterial strains. Radioactivity in the pellet, tips supernatant and micro-centrifugation tubes was counted separately for 5 s in a sodium iodide well-counter with dedicated nuclear medicine software. The overall percentage activity of the live and killed bacteria was found in the range of 5–46% with different types of labelled quinolones and bacteria. Activity of the labelled enorfloxacin and ciprofloxacin indicated acceptable results for both live and killed E. coli and Ps. aeruginosa . However, ofloxacin was found to be moderate for all the live bacterial stains. This developed methodology has achieved more than 95% labelling efficiency of 99m Tc with derivatized quinolones and also the observed results indicated that these complexes may be used as an infection specific imaging agents.