Aim: Although radiological methods are sufficient for the diagnosis of spontaneous subarachnoid hemorrhage (SAH), additional biomarkers are needed to predict prognosis. The aim of this study was to investigate the effects of serum S100B protein, Glial Fibrillary Acidic Protein (GFAP) levels and, Optic Nerve Sheath Diameter (ONSD) on mortality and clinical severity in patients with spontaneous SAH. Materials and methods: Fifty-six patients who were diagnosed with SAH after first evaluation in the emergency department (ED) were included in the study group; Forty-six patients who were admitted to the ED with headache of non-intracranial etiology, were included as the control group. Cerebral computed tomography (CT) images and peripheral blood samples were obtained from all patients; at the time of diagnosis and 24 h after diagnosis. Serum S100B protein and GFAP levels were measured from the blood samples and ONSD was measured on CT. Results: Serum S100B protein and GFAP levels and, ONSDs at the time of diagnosis and 24 h after diagnosis were significantly higher in the study group (p < 0.05). Both GFAP levels and ONSD at the time of diagnosis and 24 h after the diagnosis were found to be related with increased mortality (p < 0.05). A similar association was found for serum S100B protein levels 24 h after the diagnosis, but not at the time of diagnosis (p = 0.540). Conclusion: Serum S100B protein and GFAP levels and, ONSD were increased in patients with spontaneous SAH. All parameters were found to be associated with increased mortality.