Staphylococcus aureus is the leading cause of life-threatening infections, frequently originating from unknown or deep-seated foci. Source control and institution of appropriate antibiotics remain challenges, especially with infections due to methicillin-resistant S. aureus (MRSA). In this study, we developed a radiofluorinated analog of para-aminobenzoic acid (2-[¹⁸F]F-PABA) and demonstrate that it is an efficient alternative substrate for the S. aureus dihydropteroate synthase (DHPS). 2-[¹⁸F]F-PABA rapidly accumulated in vitro within laboratory and clinical (including MRSA) strains of S. aureus but not in mammalian cells. Biodistribution in murine and rat models demonstrated localization at infection sites and rapid renal elimination. In a rat model, 2-[¹⁸F]F-PABA positron emission tomography (PET) rapidly differentiated S. aureus infection from sterile inflammation and could also detect therapeutic failures associated with MRSA. These data suggest that 2-[¹⁸F]F-PABA has the potential for translation to humans as a rapid, noninvasive diagnostic tool to identify, localize, and monitor S. aureus infections.