The aim of this study was to analyze and compare the humoral immune responses in serum and cerebrospinal fluid (CSF) for 34 adult patients with clinically evident Lyme neuroborreliosis, 27 patients with clinically suspected Lyme neuroborreliosis, and 32 patients with tick-borne encephalitis. Additionally, we wanted to compare the findings of two methods for the detection of intrathecally synthesized borrelial antibodies: the IDEIA Lyme neuroborreliosis test using flagellar antigen and an approach based on the Liaison indirect chemiluminescence immunoassay using the OspC and VlsE antigens. Borrelial IgM and IgG antibodies were detected by at least one of these methods in the sera of 22/34 (64.7%) and 28/34 (82.4%) patients with clinically evident Lyme neuroborreliosis, respectively, and in the cerebrospinal fluid of 22/34 (64.7%) and 20/34 (58.8%) of these patients, respectively. Intrathecal synthesis of borrelial IgM and/or IgG was found in 19/34 (55.9%) patients: IgM in 17/34 (50%) patients and IgG in 15/34 (44.1%) patients. The relatively low proportion of intrathecal synthesis of borrelial antibodies and the high ratio of IgM positivity could be explained by the short duration of neurological disease as evidenced by reported symptoms (median, 10 days). Assessment of the humoral immune response in the sera and CSF of patients with early Lyme neuroborreliosis confirmed previous findings on the relationship between the duration of illness and the proportion of patients with detectable responses.