Nuclear medicine clinicians are still waiting for the optimal scintigraphic imaging agents capable of distinguishing between infection and inflammation, and between fungal and bacterial infections. Aptamers have several properties that make them suitable for molecular imaging. In the present study, a peptidoglycan aptamer (Antibac1) was labeled with ^99mTc and evaluated by biodistribution studies and scintigraphic imaging in infection‐bearing mice. Labeling with ^99mTc was performed by the direct method and the complex stability was evaluated in saline, plasma and in the molar excess of cysteine. The biodistribution and scintigraphic imaging studies with the ^99mTc-Antibac1 were carried out in two different experimental infection models: Bacterial-infected mice (S. aureus) and fungal-infected mice (C. albicans). A ^99mTc radiolabeled library, consisting of oligonucleotides with random sequences, was used as a control for both models. Radiolabeling yields were superior to 90% and ^99mTc‐Antibac1 was highly stable in presence of saline, plasma, and cysteine up to 6h. Scintigraphic images of S. aureus infected mice at 1.5 and 3.0h after ^99mTc‐Antibac1 injection showed target to non-target ratios of 4.7±0.9 and 4.6±0.1, respectively. These values were statistically higher than those achieved for the ^99mTc‐library at the same time frames (1.6±0.4 and 1.7±0.4, respectively). Noteworthy, ^99mTc‐Antibac1 and ^99mTc‐library showed similar low target to non-target ratios in the fungal-infected model: 2.0±0.3 and 2.0±0.6for ^99mTc‐Antibac1 and 2.1±0.3 and 1.9 ± 0.6 for ^99mTc‐library, at the same times. These findings suggest that the ^99mTc‐Antibac1 is a feasible imaging probe to identify a bacterial infection focus. In addition, this radiolabeled aptamer seems to be suitable in distinguishing between bacterial and fungal infection.