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ABSTRACT Although the majority of patients with Lyme disease can be cured with the standard 2-4 week antibiotic treatment, about 10-20% of patients continue to suffer from post-treatment Lyme disease syndrome (PTLDS). While the cause for this is debated, one possibility is due to persisters not killed by the current Lyme antibiotics. It has been reported that essential oils have antimicrobial activities and some have been used by patients with persisting Lyme disease symptoms. However, the activity of essential oils against the causative agent Borrelia burgdorferi (B. burgdorferi) has not been carefully studied. Here, we evaluated the activity of 34 essential oils against B. burgdorferi stationary phase culture as a model for persisters. We found that many essential oils had varying degrees of activity against B. burgdorferi , with top 5 essential oils (oregano, cinnamon bark, clove bud, citronella, and wintergreen) at a low concentration of 0.25% showing more activity than the persister drug daptomycin. Interestingly, some highly active essential oils were found to have excellent anti-biofilm ability as shown by their ability to dissolve the aggregated biofilm-like structures. The top 3 hits, oregano, cinnamon bark and clove bud, completely eradicated all viable cells without regrowth in subculture. Carvacrol was found to be the most active ingredient of oregano oil showing excellent activity against B. burgdorferi stationary phase cells, while p-cymene and α-terpinene had no apparent activity. Future studies are needed to characterize and optimize the active essential oils in drug combinations in vitro and in vivo for improved treatment of persistent Lyme disease. IMPORTANCE There is a huge need for effective treatment of patients with Lyme disease who suffer from PTLDS. Recent in vitro and in vivo studies suggest that B. burgdorferi develops persisters that are not killed by the current Lyme antibiotics as a possible contributor to this condition. Although essential oils are used by patients with Lyme disease with variable improvement in symptoms, their anti-borrelia activity has not been carefully studied. Here we found that not all essential oils have adequate anti-borrelia activity and identified some highly potent essential oils (oregano, cinnamon bark, clove bud) that have even higher anti-persister and anti-biofilm activity than the persister drug daptomycin. Carvacrol was found to be the most active ingredient of oregano oil and have the potential to serve as a promising oral persister drug. Our findings may have implications for developing improved treatment of persisting Lyme disease.