Remarkably, we found that daptomycin in combination with GGsTop doxycycline and GKA 50 cefoperazone was able to completely eradicate the most resistant microcolonies, and this was further confirmed by subculture studies which showed lack of any growth. While various drug combinations showed improved activity against stationary phase B. burgdorferi persisters, daptomycin combinations had the best activity among drug combinations against persisters. The only non-daptomycin regimens that were close to daptomycin combinations contained cefoperazone. Unexpectedly, other antibiotics such as sulfamethoxazole, clofazimine and miconazole also had more activity against stationary phase B. burgdorferi persisters in combination with doxycycline and cefoperazone. These drugs are not currently used as antibiotics for treatment of Lyme disease clinically. Although sulfa drugs are bacteriostatic when used alone for growing bacteria, they could kill non-growing round body or aggregated microcolony form of B. burgdorferi during long-term treatment. Clofazimine with high anti-persister activity improved the combination with daptomycin or daptomycin plus doxycycline which may be due to its multiple mechanisms of action including membrane destabilization, reactive oxygen species production, and inhibition of membrane energy metabolism in M. tuberculosis. We also found that miconazole, an imidazole antifungal drug, had high activity against B. burgdorferi persisters when combined with doxycycline and cefoperazone. Miconazole has been shown to alter the integrity of lipid membrane and therefore may facilitate the penetration of other drugs such as doxycycline and cefoperazone for improved activity against B. burgdorferi persisters. The complete eradication of the B. burgdorferi biofilm-like microcolonies by the three drug combination of daptomycin+doxycycline+cefoperazone has not been achieved with any single, dual or even some three drug combinations in this study or any other previous studies. The mechanism by which this three drug combination was able to achieve this remarkable activity is worth commenting. Doxycycline and cefoperazone inhibits protein synthesis and cell wall peptidoglycan synthesis respectively. Either may be needed to kill the growing forms present in the B. burgdorferi microcolonies or those occasionally revert to growing forms from microcolonies, but these drugs are less effective against the round body or microcolony persisters themselves. This inability could be because of the reduced drug penetration into the microcolony structure, efflux mechanism, or decreased protein or cell wall synthesis in persisters.