It can be difficult to establish whether a febrile episode in a patient is suggestive of an infectious or non-infectious cause. Besides clinical history, physical examination, and laboratory assays, scintigraphic imaging of bacterial and fungal infections using antimicrobial peptides labelled with technetium-99m (99mTc) can be useful. Key to this latter approach is that some of these peptides accumulate at sites of infection but not in sterile inflammatory lesions, because of their preferential binding to bacteria and fungi over mammalian cells. Here we report on imaging of infections with these peptides in laboratory animals. On the basis of their favourable binding characteristics, fast and easy penetration into the infected area, and rapid clearance from the circulation (half-life approximately 30 min) via the kidneys, several 99mTc-antimicrobial peptides have been selected that distinguish infectious foci from sites of sterile inflammation. Accumulation of 99mTc-antimicrobial peptides at sites of experimental infection correlated well with the number of viable bacteria/yeasts present. This finding allowed us to monitor with 99mTc-antimicrobial peptides the efficacy of antimicrobial therapy in animals with experimental infections. In conclusion, non-microbicidal amounts of 99mTc-antimicrobial peptides are promising candidates for the scintigraphic imaging of bacterial/fungal infections and for monitoring the efficacy of antimicrobial therapy in patients.