Enterobacterales represent the largest group of bacterial pathogens in humans and are responsible for severe, deep-seated infections, often resulting in sepsis or death. They are also a prominent cause of multidrug-resistant (MDR) infections, and some species are recognized as biothreat pathogens. Tools for noninvasive, whole-body analysis that can localize a pathogen with specificity are needed, but no such technology currently exists. We previously demonstrated that positron emission tomography (PET) with 2-deoxy-2-[¹⁸F]fluoro-d-sorbitol (¹⁸F-FDS) can selectively detect Enterobacterales infections in murine models. Here, we demonstrate that uptake of ¹⁸F-FDS by bacteria occurs via a metabolically conserved sorbitol-specific pathway with rapid in vitro ¹⁸F-FDS uptake noted in clinical strains, including MDR isolates. Whole-body ¹⁸F-FDS PET/computerized tomography (CT) in 26 prospectively enrolled patients with either microbiologically confirmed Enterobacterales infection or other pathologies demonstrated that ¹⁸F-FDS PET/CT was safe, could rapidly detect and localize Enterobacterales infections due to drug-susceptible or MDR strains, and differentiated them from sterile inflammation or cancerous lesions. Repeat imaging in the same patients monitored antibiotic efficacy with decreases in PET signal correlating with clinical improvement. To facilitate the use of ¹⁸F-FDS, we developed a self-contained, solid-phase cartridge to rapidly (<10 min) formulate ready-to-use ¹⁸F-FDS from commercially available 2-deoxy-2-[¹⁸F]fluoro-d-glucose (¹⁸F-FDG) at room temperature. In a hamster model, ¹⁸F-FDS PET/CT also differentiated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia from secondary Klebsiella pneumoniae pneumonia—a leading cause of complications in hospitalized patients with COVID-19. These data support ¹⁸F-FDS as an innovative and readily available, pathogen-specific PET technology with clinical applications.