Opportunistic infections are a problem of great relevance in public health and the precise detection and localization of infection in the early stages of the disease is of great importance for patient management as well as cost containment. Our proposal seeks to contribute to developing a new agent that meets the needs of diagnosis and follow-up of fungal and bacterial infections, focused on the design of a radiotracer with the potential for recognition of hidden infection foci. Defensins are plant antimicrobial peptides that not only show activity against plant pathogens but also against human ones. A short analogue of EcgDf1 defensin, EcgDf21d (NH2-ERFTGGHCRGFRRRCFCTKHC-COOH), was labelled through the formation of a 99mTc-HYNIC complex which was assessed for physicochemical and biological behaviour both in vitro and in vivo. The [99mTc]Tc-HYNIC-EcgDf21 labelling procedure rendered a single product with remarkably high RCP and stability in the labelling milieu. The Log p value indicated that [99mTc]Tc-HYNIC-EcgDf21 has a hydrophilic behaviour, confirmed by the biodistribution profiles. The optimal uptake value was obtained for Candida albicans infection model reaching a lesion/muscle ratio of 3, this correlates with in vitro binding studies, and the lesion can be definitely observed in the scintigraphic images.