<title>Abstract</title>Addressing the growing problem of antibiotic resistance requires the development of new drugs with novel antibacterial targets. FtsZ has been identified as an appealing new target for antibacterial agents. Here, we describe the structure-guided design of a new fluorescent probe (<bold>BOFP</bold>) in which a BODIPY fluorophore has been conjugated to an oxazole-benzamide FtsZ inhibitor. Crystallographic studies have enabled us to identify the optimal position for tethering the fluorophore that facilitates the high-affinity FtsZ binding of <bold>BOFP</bold>. Fluorescence anisotropy studies demonstrate that <bold>BOFP</bold> binds the FtsZ proteins from the Gram-positive pathogens <italic>Staphylococcus aureus</italic>, <italic>Enterococcus faecalis</italic>, <italic>Enterococcus faecium</italic>, <italic>Streptococcus pyogenes</italic>, <italic>Streptococcus agalactiae</italic>, and <italic>Streptococcus pneumoniae</italic> with K_d values of 0.6–4.6 µM. Significantly, <bold>BOFP</bold> binds the FtsZ proteins from the Gram-negative pathogens <italic>Escherichia coli</italic>, <italic>Klebsiella pneumoniae</italic>, <italic>Pseudomonas aeruginosa</italic>, and <italic>Acinetobacter baumannii</italic> with an even higher affinity (K_d = 0.2–0.8 µM). Fluorescence microscopy studies reveal that <bold>BOFP</bold> can effectively label FtsZ in all the above Gram-positive and Gram-negative pathogens. In addition, <bold>BOFP</bold> is effective at monitoring the impact of non-fluorescent inhibitors on FtsZ localization in these target pathogens. Viewed as a whole, our results highlight the utility of <bold>BOFP</bold> as a powerful tool for identifying new broad-spectrum FtsZ inhibitors and understanding their mechanisms of action.