Background: Biochemical markers of brain pathology could potentially contribute to diagnosis and prediction in epilepsy. We describe levels of five brain injury markers in adults with new-onset seizures, and assess group differences in patients with a single seizure, epilepsy, and poststroke epilepsy. Methods: In this prospective observational study, adults with new-onset seizures were recruited at Sahlgrenska University Hospital, Sweden, and concentrations of glial fibrillary acidic protein (GFAP), neurofilament light (NfL), microtubule-associated protein tau (tau), S100 calcium-binding protein (S100B), and neuron-specific enolase (NSE) were measured. Participants were categorized as epilepsy, poststroke epilepsy (PSE), or single seizure (no additional seizures). Patients were followed until a diagnosis of epilepsy or PSE, or for at least two years in single seizure cases. Results: The cohort included 23 (37%) individuals with a single seizure, 24 (39%) with epilepsy, and 15 (24%) with PSE. The concentrations of S100B were higher in patients with epilepsy and PSE than in single seizures (p = 0.0023 and p = 0.0162, respectively). The concentrations of NfL were higher in patients with PSE than in single seizures (p=0.0027). After age-normalization, levels of S100B were higher in patients with epilepsy and levels of NfL were higher in patients with PSE (p = 0.0021 and p = 0.0180). Conclusion: Levels of S100B and NfL were higher in patients with epilepsy or PSE than patients with single seizures. Further studies are needed to investigate the biomarker potential of brain injury markers as predictors of epilepsy course or indicators of epileptogenesis.