Lyme disease (LD), caused by Borrelia burgdorferi, is the most common vector-borne disease in USA and Europe. Despite the standard 2-4 weeks antibiotic treatment, approximately 10%-20% of patients will develop post-treatment LD syndrome, a condition that is poorly understood. One of the probable causes is thought to be the presence of B. burgdorferi persister forms that are not effectively killed by the current LD antibiotics. In this study, we evaluated nitroxoline, an antibiotic used to treat urinary tract infections, for its activity against a stationary-phase culture enriched with persister forms of B. burgdorferi. Nitroxoline was found to be more active than doxycycline and equally active as cefuroxime (standard LD antibiotics) against B. burgdorferi. Importantly, the nitroxoline two-drug combinations nitroxoline + cefuroxime and nitroxoline + clarithromycin, as well as the nitroxoline three-drug combination nitroxoline + cefuroxime + clarithromycin were as effective as the persister drug daptomycin-based positive control three-drug combination cefuroxime + doxycycline + daptomycin, completely eradicating stationary-phase B. burgdorferi in the drug-exposure experiments, and preventing regrowth in the subculture study. Future studies should evaluate these promising drug combinations in a persistent LD mouse model.