Background and purpose <p>This study aimed to elucidate the differences between patients with anti-glial fibrillary acidic protein (GFAP) antibodies who exhibited concurrent positivity for other neuro-antibodies and those who did not.</p> Methods <p>Hospitalised patients demonstrating anti-GFAP antibody positivity in cerebrospinal fluid (CSF) were retrospectively collected and followed up from March 2019 to July 2022. Data including clinical features, laboratory results, imaging findings, therapy, and prognosis were extracted from the medical record system.</p> Results <p>Thirty-seven patients with positive anti-GFAP antibody in CSF were included. Ten patients exhibited concomitant other neuro-antibodies and were categorised as the “overlapping group”. Compared to the non-overlapping group, the incidence of seizures was significantly higher in the overlapping syndrome group (50% vs. 3.7%, <i>p</i> = 0.003), and a similar trend was observed for status epilepticus (30% vs. 3.7%, <i>p</i> = 0.052). Encephalitis was more prevalent in the overlapping group (60.0%) than in the non-overlapping group (11.1%) (<i>P</i> = 0.005). Serum white blood cell and neutrophil counts were significantly higher in the overlapping group compared to the non-overlapping group (<i>P</i> = 0.037, <i>P</i> = 0.042, respectively). The overlapping group demonstrated a higher percentage of immunosuppressant usage and a longer hospital stay compared to the non-overlapping group. During the follow-up, two patients in the overlapping group expired, while all the patients in the non-overlapping group survived.</p> Conclusions <p>Patients with anti-GFAP antibodies and concurrent other neuro-antibodies exhibited different clinical features compared to non-overlapping cases. We suggest that in the presence of coexisting neuronal surface antibodies, these specific neuro-antibodies were predominant in clinical manifestations and prognosis, rather than the anti-GFAP antibody, especially when in a relatively high titer.</p>