In this study, 99mTc–plazomicin, a new radio-antibiotic complex, was prepared specifically for bacterial infection localization and monitoring. Factors affecting the labeling reaction were studied and optimized to obtain a high yield (98.8 ± 0.2%). In-silico, radiochemical and physicochemical characterization and biodistribution were performed to assess the complex aptness as a radiopharmaceutical. The complex was biologically evaluated in-vitro using bacteria and in-vivo using different inflammation models (sterile, bacterial, and fungal). Uptake in the bacterial model was highest (7.8 ± 0.3%). Results indicated that the technetium label did not alter the antibiotic biological behavior and backed the usefulness of 99mTc-plazomicin as a potential tracer.