Lyme disease, the most common vector-borne zoonosis in North America, is caused by the spirochetal pathogen Borrelia burgdorferi. The telomere resolvase encoded by this organism (ResT) promotes the formation of covalently closed hairpin ends on the linear DNA molecules of B. burgdorferi through a two-step transesterification. ResT is essential for survival and is therefore an attractive target for the development of highly specific antiborrelial drugs. To identify ResT inhibitors, a novel fluorescence-based high-throughput assay was developed and used to screen a library of 27,520 small-molecule drug-like compounds. Six confirmed inhibitors of ResT, with 50% inhibitory concentrations between 2 and 10 muM, were identified. The inhibitors were characterized further and were grouped into three distinct classes based on their inhibitory features. The high-throughput screening assay developed in this paper, along with the six inhibitory compounds identified, provides a starting point for the future development of novel antiborrelial drugs as well as small-molecule inhibitors that will be helpful for the further dissection of the reaction mechanism.