Human Lyme borreliosis is a multisystem disorder that can progress in stages and is transmitted by ticks of the Ixodes ricinus complex infected with the spirochete Borrelia burgdorferi sensu lato. Today, Lyme borreliosis is regarded as the most important human tickborne illness in the northern hemisphere. Soon after the causative agent was correctly identified and successfully isolated in 1982, antibiotic treatment was shown to be effective and since then a variety of in vitro and in vivo studies have been performed to further characterize the activity of antimicrobial agents against B. burgdorferi s.l. Although many antimicrobial agents have been tested for their in vitro activity against borreliae, the full spectrum of antibiotic susceptibility in B. burgdorferi s.l. has not been defined for many compounds. Moreover, our current understanding of possible antimicrobial resistance mechanisms in B. burgdorferi s.l. is limited and is largely founded on in vitro experiments on relatively few borrelial isolates. This review will summarize what is and what is not known about antimicrobial resistance in B. burgdorferi s.l. and will discuss open questions that continue to fuel the current debate on treatment-resistant Lyme borreliosis.