Abstract Purpose With the increase of especially hospital-acquired infections, timely and accurate diagnosis of bacterial infections is crucial for effective patient care. Molecular imaging has the potential for specific and sensitive detection of infections. Siderophores are iron-specific chelators recognized by specific bacterial transporters, representing one of few fundamental differences between bacterial and mammalian cells. Replacing iron by gallium-68 without loss of bioactivity is possible allowing molecular imaging by positron emission tomography (PET). Here, we report on the preclinical evaluation of the clinically used siderophore, desferrioxamine-B (Desferal®, DFO-B), radiolabelled with ⁶⁸Ga for imaging of bacterial infections. Methods In vitro characterization of [⁶⁸Ga]Ga-DFO-B included partition coefficient, protein binding and stability determination. Specific uptake of [⁶⁸Ga]Ga-DFO-B was tested in vitro in different microbial cultures. In vivo biodistribution was studied in healthy mice and dosimetric estimation for human setting performed. PET/CT imaging was carried out in animal infection models, representing the most common pathogens. Results DFO-B was labelled with ⁶⁸Ga with high radiochemical purity and displayed hydrophilic properties, low protein binding and high stability in human serum and PBS. The high in vitro uptake of [⁶⁸Ga]Ga-DFO-B in selected strains of Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus agalactiae could be blocked with an excess of iron-DFO-B. [⁶⁸Ga]Ga-DFO-B showed rapid renal excretion and minimal retention in blood and other organs in healthy mice. Estimated human absorbed dose was 0.02 mSv/MBq. PET/CT images of animal infection models displayed high and specific accumulation of [⁶⁸Ga]Ga-DFO-B in both P. aeruginosa and S. aureus infections with excellent image contrast. No uptake was found in sterile inflammation, heat-inactivated P. aeruginosa or S. aureus and Escherichia coli lacking DFO-B transporters. Conclusion DFO-B can be easily radiolabelled with ⁶⁸Ga and displayed suitable in vitro characteristics and excellent pharmacokinetics in mice. The high and specific uptake of [⁶⁸Ga]Ga-DFO-B by P. aeruginosa and S. aureus was confirmed both in vitro and in vivo, proving the potential of [⁶⁸Ga]Ga-DFO-B for specific imaging of bacterial infections. As DFO-B is used in clinic for many years and the estimated radiation dose is lower than for other ⁶⁸Ga-labelled radiopharmaceuticals, we believe that [⁶⁸Ga]Ga-DFO-B has a great potential for clinical translation.