In vivo diagnostic imaging of bacterial infections is currently reliant on targeting their metabolic pathways, an ineffective method to identify microbial species with low metabolic activity. Here we establish HS-198 as a small molecule-fluorescent conjugate that selectively targets the highly conserved bacterial protein, HtpG (High temperature protein G), within B. burgdorferi, the bacteria responsible for Lyme Disease. We describe the use of HS-198 to target morphologic forms of B. burgdorferi in both the logarithmic growth phase and the metabolically dormant stationary phase. Furthermore, in a murine infection model, systemically injected HS-198 identified B. burgdorferi as revealed by imaging in post necropsy tissue sections. These findings demonstrate how small molecule probes directed at conserved bacterial protein targets can function to identify the microbe using non-invasive imaging and potentially as scaffolds to deliver antimicrobial agents to the pathogen.