AimsBorrelia sp., a causative pathogenic factor of Lyme disease (LD), has become a major public health threat. Current treatments based on antibiotics often lead to relapse after their withdrawal. Naturally derived substances that could work synergistically to display higher efficacy compared with the individual components may serve as a resource for the development of novel approaches to combat both active and latent forms of Borrelia sp. Methods and ResultsUsing checkerboard assay, we investigated the anti-borreliae reciprocal cooperation of phytochemicals and micronutrients against two species of Borrelia selected as prevalent causes of LD in the United States and Europe. We tested 28 combinations of phytochemicals such as polyphenols (baicalein, luteolin, rosmarinic acids), fatty acids (monolaurin, cis-2-decenoic acid) and micronutrients (ascorbic acid, cholecalciferol and iodine). The results showed that the combinations of baicalein with luteolin as well as monolaurin with cis-2-decenoic acid expressed synergistic anti-spirochetal effects. Moreover, baicalein and luteolin, when combined with rosmarinic acid or iodine, produced additive bacteriostatic and bactericidal effects against typical corkscrew motile spirochaetes and persistent knob/round-shaped forms, respectively. An additive anti-biofilm effect was noticed between baicalein with luteolin and monolaurin with cis-2-decenoic acid. Finally, application of the combination of baicalein with luteolin increased cytoplasmic permeability of Borrelia sp. but did not cause DNA damage. ConclusionsThese results show that a specific combination of flavones might play a supporting role in combating Borrelia sp. through either synergistic or additive anti-borreliae effects. Significance and Impact of the StudyPresented here invitro results might help advancing our knowledge and improving the approach to target Borrelia sp.