Deep-seated bacterial infections are difficult to detect and diagnose due to the lack of specific clinical imaging modalities. Therefore, the bacteria-specific positron emission tomography radiotracer 2-[18F]fluoro-4-nitrobenzoic acid ([18F]FNB) was developed, which is reduced to 2-[18F]fluoro-4-aminobenzoic acid ([18F]F-PABA) by bacterial nitroreductases and has improved pharmacokinetics compared to the parent compound. PET imaging demonstrated that the uptake of 2-[18F]fluoro-4-nitrobenzoic acid in a clinically relevant Staphylococcus aureus prosthetic joint infection model was up to ∼4-fold higher in the infected joint compared to the contralateral joint. 2-[18F]Fluoro-4-nitrobenzoic acid was also able to distinguish infection from inflammation in a surgical inflammation model. Based on the mouse radiation dosimetry results, the calculated effective dose of 2-[18F]fluoro-4-nitrobenzoic acid was well below the whole-body radiation dose limit established by the Food and Drug Administration for humans. In addition, no treatment-related microscopic changes in organ histopathology were observed in a mouse acute toxicity study. Overall, these data suggest that 2-[18F]fluoro-4-nitrobenzoic acid is a specific and effective imaging agent for noninvasively diagnosing prosthetic joint infections.