Neuroborreliosis (NB) is a chronic infectious disease of the central nervous system (CNS) caused by a tick-borne spirochete, Borrelia burgdorferi. In addition to direct effects of the causative infectious agent, additional immunity-mediated mechanisms are thought to play a role in the CNS pathology of NB. In order to further understand the involvement of humoral immune mechanisms in NB, we dissected the intrathecal antibody responses down to the single-plasma-cell level. Starting with single-cell reverse transcription-PCR of fluorescence-activated cell sorter-sorted cerebrospinal fluid plasma cells from an NB patient, we identified expanded clones and resurrected the antigen specificity of their secreted antibodies through recombinant expression of the correctly paired immunoglobulin heavy- and light-chain genes as monoclonal antibodies (MAbs). As expected, we found specificity for the causative infectious agent, B. burgdorferi, among the clonally expanded plasma cell (cePC)-derived MAbs. However, from an independent cePC of the same patient, we could derive MAbs specific for human CNS myelin, without detectable cross-reactivity with B. burgdorferi antigens. While reactivity against B. burgdorferi is a known feature of humoral immune responses in NB, we show (i) that immune responses specific for self antigens may be a distinct feature of CNS infections independent of pathogen reactivity and (ii) that humoral autoimmunity in NB (since found in cePC) is the result of a truly antigen-driven immune response. Our findings indicate that in NB mechanisms may be at play that induce distinct immune responses specific for pathogen and self antigens independent from “molecular mimicry.”