There is often overlap in the diagnostic features of common pathologic processes such as infection, sterile inflammation, and cancer both clinically and using conventional imaging techniques. Here, we report the development of a positron emission tomography probe for live bacterial infection based on the small-molecule antibiotic trimethoprim (TMP). [¹⁸F]fluoropropyl-trimethoprim, or [¹⁸F]FPTMP, shows a greater than 100-fold increased uptake in vitro in live bacteria (Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa) relative to controls. In a rodent myositis model, [¹⁸F]FPTMP identified live bacterial infection without demonstrating confounding increased signal in the same animal from other etiologies including chemical inflammation (turpentine) and cancer (breast carcinoma). Additionally, the biodistribution of [¹⁸F]FPTMP in a nonhuman primate shows low background in many important tissues that may be sites of infection such as the lungs and soft tissues. These results suggest that [¹⁸F]FPTMP could be a broadly useful agent for the sensitive and specific imaging of bacterial infection with strong translational potential.