Lyme disease is the leading tick-borne disease in the USA. Whereas the majority of Lyme disease patients with early disease can be cured with standard treatment, some patients suffer from chronic fatigue and joint and muscular pain despite treatment, a syndrome called posttreatment Lyme disease syndrome. Although the cause is unclear, ineffective killing of Borrelia burgdorferi persisters by current Lyme disease antibiotics is one possible explanation. We took advantage of our recently developed high-throughput viability assay and screened the National Cancer Institute compound library collection consisting of 2526 compounds against stationary phase B. burgdorferi. We identified the top 30 new active hits, including the top six anthracycline antibiotics daunomycin 3-oxime, dimethyldaunomycin, daunomycin, NSC299187, NSC363998 and nogalamycin, along with other compounds, including prodigiosin, mitomycin, nanaomycin and dactinomycin, as having excellent activity against B. burgdorferi stationary phase culture. The anthracycline or anthraquinone compounds, which are known to have both anti-cancer and antibacterial activities, also had high activity against growing B. burgdorferi with low minimum inhibitory concentration. Future studies on the structure-activity relationship and mechanisms of action of anthracyclines/anthraquinones are warranted. In addition, drug combination studies with the anthracycline class of compounds and the current Lyme antibiotics to eradicate B. burgdorferi persisters in vitro and in animal models are needed to determine if they improve the treatment of Lyme disease.