The Enterobacteriaceae are a family of rod-shaped Gram-negative bacteria that normally inhabit the gastrointestinal tract and are the most common cause of Gram-negative bacterial infections in humans. In addition to causing serious multidrug-resistant, hospital-acquired infections, a number of Enterobacteriaceae species are also recognized as biothreat pathogens. As a consequence, new tools are urgently needed to specifically identify and localize infections due to Enterobacteriaceae and to monitor antimicrobial efficacy. In this report, we used commercially available 2-[¹⁸F]-fluorodeoxyglucose (¹⁸F-FDG) to produce 2-[¹⁸F]-fluorodeoxysorbitol (¹⁸F-FDS), a radioactive probe for Enterobacteriaceae, in 30 min. ¹⁸F-FDS selectively accumulated in Enterobacteriaceae, but not in Gram-positive bacteria or healthy mammalian or cancer cells in vitro. In a murine myositis model, ¹⁸F-FDS positron emission tomography (PET) rapidly differentiated true infection from sterile inflammation with a limit of detection of 6.2 ± 0.2 log₁₀ colony-forming units (CFU) for Escherichia coli. Our findings were extended to models of mixed Gram-positive and Gram-negative thigh co-infections, brain infection, Klebsiella pneumonia, and mice undergoing immunosuppressive chemotherapy. This technique rapidly and specifically localized infections due to Enterobacteriaceae, providing a three-dimensional holistic view within the animal. Last, ¹⁸F-FDS PET monitored the efficacy of antimicrobial treatment, demonstrating a PET signal proportionate to the bacterial burden. Therapeutic failures associated with multidrug-resistant, extended-spectrum β-lactamase (ESBL)–producing E. coli infections were detected in real time. Together, these data show that ¹⁸F-FDS is a candidate imaging probe for translation to human clinical cases of known or suspected infections owing to Enterobacteriaceae.