More than 40 human cases of severe encephalitis caused by Borna disease virus 1 (BoDV-1) have been reported to German health authorities. In an endemic region in southern Germany, we conducted the seroepidemiological BoSOT study (“BoDV-1 after solid-organ transplantation”) to assess whether there are undetected oligo- or asymptomatic courses of infection. A total of 216 healthy blood donors and 280 outpatients after solid organ transplantation were screened by a recombinant BoDV-1 ELISA followed by an indirect immunofluorescence assay (iIFA) as confirmatory test. For comparison, 288 serum and 258 cerebrospinal fluid (CSF) samples with a request for tick-borne encephalitis (TBE) diagnostics were analyzed for BoDV-1 infections. ELISA screening reactivity rates ranged from 3.5% to 18.6% depending on the cohort and the used ELISA antigen, but only one sample of a patient from the cohort with requested TBE diagnostics was confirmed to be positive for anti-BoDV-1-IgG by iIFA. In addition, the corresponding CSF sample of this patient with a three-week history of severe neurological disease tested positive for BoDV-1 RNA. Due to the iIFA results, all other results were interpreted as false-reactive in the ELISA screening. By linear serological epitope mapping, cross-reactions with human and bacterial proteins were identified as possible underlying mechanism for the false-reactive ELISA screening results. In conclusion, no oligo- or asymptomatic infections were detected in the studied cohorts. Serological tests based on a single recombinant BoDV-1 antigen should be interpreted with caution, and an iIFA should always be performed in addition.